Abstract:

In quantitation without using the isotope labeling technique, there is no
means to detect the presence/absence and the time region of the
occurrence of quantitative analysis-inhibitory factors in data for the
analysis, and the reliability of the data for the analysis cannot be
evaluated. Also, the error of the data due to the occurrence of the
quantitative analysis-inhibitory factors cannot be evaluated. In order to
solve the problems, first, an internal standard to be detected
simultaneously with a component for the analysis is mixed in a mobile
phase or an eluate of a liquid chromatograph; under the condition where
no quantitative analysis-inhibitory factors occur, a blank sample is
analyzed to acquire a mass chromatogram of ions originated from the
internal standard; and the result is stored in a data storage unit. Then,
a sample for the analysis is mixed to acquire data for the analysis of
the sample; and the intensity of ions originated from the internal
standard is compared with that of the blank sample in the analysis real
time in a data analysis unit. At this time, if an inconsistency exceeding
a predetermined threshold is detected, the occurrence of the quantitative
analysis-inhibitory factors can be detected. Further, based on the
inconsistency, the error range of the data can be given to a data set and
the like.

Claims:

1. A method of an analysis using a liquid chromatograph/mass
spectrometer, comprising the steps of: mixing a standard molecule in a
solution for the analysis; acquiring a first mass chromatogram of ions
originated from the standard molecule in a condition that mixing of a
sample for the analysis in the solution for the analysis is negligible;
acquiring a second mass chromatogram of ions originated from the standard
molecule in a condition that the sample for the analysis is mixed in the
solution for the analysis; performing a level adjustment of the first and
the second mass chromatograms; calculating an inconsistency between the
first and the second mass chromatograms, and comparing the inconsistency
with a threshold of an inconsistency stored in advance; detecting a time
region for the analysis in a condition that the inconsistency is smaller
than the threshold of an inconsistency; and collecting data for the
analysis of the sample for the analysis acquired in the time region for
the analysis, wherein a height of hydrophobicity of the standard molecule
mixed in the solution for the analysis is changed according to a ratio of
an organic solvent in a mobile phase of the liquid chromatograph.

2. The method of an analysis according to claim 1, wherein the standard
molecule is hydrophilic in the case where a ratio of the organic solvent
in the mobile phase is 50% or less.

3. The method of an analysis according to claim 1, wherein the standard
molecule is hydrophobic in the case where a ratio of the organic solvent
in the mobile phase is 70% or more.

4. The method of an analysis according to claim 1, wherein the standard
molecule has an isoelectric point or a dissociation constant of
approximately 2 or more and 8 or less.

5. The method of an analysis according to claim 1, wherein the standard
molecule has an isoelectric point or a dissociation constant of
approximately 8 or more.

6. A method of an analysis using a liquid chromatograph/mass
spectrometer, comprising the steps of: mixing a standard molecule having
an isoelectric point or a dissociation constant of approximately 2 or
more and 8 or less and a standard molecule having that of 8 or more in a
solution for the analysis; acquiring a first mass chromatogram of ions
originated from the standard molecules in a condition that mixing of a
sample for the analysis in the solution for the analysis is negligible;
acquiring a second mass chromatogram of ions originated from the standard
molecules in a condition that the sample for the analysis is mixed in the
solution for the analysis; performing a level adjustment of the first and
the second mass chromatograms; calculating an inconsistency between the
first and the second mass chromatograms, and comparing the inconsistency
with a threshold of an inconsistency stored in advance; detecting a time
region for the analysis in a condition that the inconsistency is smaller
than the threshold of an inconsistency; and collecting data for the
analysis of the sample for the analysis acquired in the time region for
the analysis, wherein a positive ion detection mode and a negative ion
detection mode is switched in one time of liquid chromatograph/mass
spectrometry.

7. A method of an analysis using a liquid chromatograph/mass
spectrometer, comprising the steps of: mixing a hydrophilic standard
molecule and a hydrophobic standard molecule in a solution for the
analysis; acquiring a first mass chromatogram of ions originated from the
hydrophilic and the hydrophobic standard molecules in a condition that
mixing of a sample for the analysis in the solution for the analysis is
negligible; acquiring a second mass chromatogram of ions originated from
the hydrophilic and the hydrophobic standard molecules in a condition
that the sample for the analysis is mixed in the solution for the
analysis; performing a level adjustment of the first mass chromatogram
and the second mass chromatogram of ions originated from the hydrophilic
standard molecule and the hydrophobic standard molecule; calculating an
inconsistency between the first and the second mass chromatograms, and
comparing the inconsistency with a threshold of an inconsistency stored
in advance; detecting a time region for the analysis in a condition that
the inconsistency is smaller than the threshold of an inconsistency; and
collecting data for the analysis of the sample for the analysis acquired
in the time region for the analysis.

8. The method of an analysis according to any one of claims 1 to 7,
comprising acquiring data of an ion peak as information regarding a peak
area of the peak or an ion intensity thereof and an error thereof, a
retention time, and m/z or m, or m and z of the peak, based on the data
of the sample for the analysis.

9. The method of an analysis according to any one of claims 1 to 7,
comprising acquiring data of an ion peak as information regarding a peak
area of the peak or an ion intensity thereof, the presence/absence of the
occurrence of a quantitative analysis-inhibitory factor, a retention
time, and m/z or m, or m and z of the peak, based on the data of the
sample for the analysis.

10. The method of a mass analysis according to claim 8 or 9, comprising
comparing the information acquired from a plurality of the samples for
the analysis.

11. An internal standard, wherein the internal standard is an internal
standard used to detect a quantitative analysis-inhibitory factor in an
analysis of positive ions using a liquid chromatograph/mass spectrometer,
and is acidic.

12. The internal standard according to claim 11, wherein the internal
standard has an isoelectric point or a dissociation constant of 8 or
less.

13. The internal standard according to claim 11, wherein the internal
standard has an isoelectric point or a dissociation constant of 4 or
less.

14. An internal standard, wherein the internal standard is an internal
standard used to detect a quantitative analysis-inhibitory factor in an
analysis of negative ions using a liquid chromatograph/mass spectrometer,
and is basic.

15. The internal standard according to claim 14, wherein the internal
standard has an isoelectric point or a dissociation constant of 8 or
more.

16. A method of an analysis using an internal standard according to claim
11 or 14.

17. A method of an analysis of a solution for the analysis containing an
object substance for the analysis by using a sample preparation unit, an
ionization unit, a mass-analysis unit, a control unit and a storage unit,
comprising: a step of mixing an internal standard in the solution for the
analysis; a step of introducing the solution for the analysis mixed with
the internal standard to the ionization unit to produce ions; a first
step of measuring an intensity of ions originated from the internal
standard by the mass-analysis unit in a condition that the internal
standard is mixed in the solution for the analysis containing a constant
or less concentration of the object substance for the analysis, and
storing a result thereof in the storage unit; a second step of measuring
intensities of ions originated from the object substance for the analysis
and the internal standard by the mass-analysis unit in a condition that
the internal standard is mixed in the solution for the analysis
containing an unknown concentration of the object substance for the
analysis, and storing results thereof in the storage unit; a step of
calculating an inconsistency between the intensities of ions originated
from the internal standard measured in the first and the second steps,
and comparing the difference with a threshold of an inconsistency stored
in advance in the storage unit, in the control unit; a step of judging
weather or not the difference exceeds the threshold of an inconsistency
in the control unit; and a step of changing the analysis condition of the
solution for the analysis in the sample preparation unit, remeasuring the
solution for the analysis containing the object substance for the
analysis, and calculating a quantitative value of the object substance
for the analysis, in the control unit, depending on the judgment.

18. The method of an analysis according to claim 17, wherein the internal
standard comprises a first internal standard and a second internal
standard, and both of an ion intensity of the first internal standard and
an ion intensity of the second internal standard are used for the
threshold and the judgment.

19. The method of an analysis according to claim 18, wherein the second
internal standard has an isoelectric point or a dissociation constant of
approximately 2 or more and 8 or less.

20. The method of an analysis of the object substance for the analysis
according to claim 18, wherein by using the first internal standard as a
quantitative internal standard for the quantitative value correction of a
measurement value of the quantitative analysis of the object substance
for the analysis, and by using the second internal standard for the
threshold and the judgment, a precision in the quantitative correction by
the first internal standard molecule is guaranteed.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a mass spectrometry system for
organism-related substances and organic substances, and the like, and
particularly to a quantitative mass spectrometry and a mass spectrometry
system for pharmacokinetics and drug metabolism in drug discovery,
protein analyses, and searches for clinical markers. The present
invention relates further to an automatic tester and an automatic
analyzer to analyze body fluids and the like.

BACKGROUND ART

[0002] In marker searches and the like for disease diagnoses, it is
important that analysis results of samples originated from disease
subjects and samples originated from healthy subjects be compared and
that variant components exhibiting outstanding differences be extracted
from among detected components. Additionally, it is also necessary that
the variant components be identified. In such analyses, liquid
chromatograph/mass spectrometers (LC/MS) are often used. The LC/MS is an
on-line system capable of separating a sample containing multiple
components by LC and analyzing the masses of separated components by MS,
and so it is used broadly also in the fields other than marker searches.
For the mass spectrometer (MS) unit, a mass spectrometer having a high
mass resolving power and securing three or more digits in the dynamic
range is often used which can perform the tandem mass spectrometry such
as MS/MS analysis. Thereby, a series of analyses can precisely be
performed which includes the extraction of variant components by the
quantitation, the identification (qualitative analysis) of a large number
of unknown components in variant components, and the measurement of the
contents thereof by the quantitative analysis.

[0003] As well known, the tandem mass spectrometry is a technology in
which ions of a component are selected from a result of a mass
spectrometry, and made to impact against a gas molecule, or otherwise, to
be decomposed, and ions produced by the decomposition are further
subjected to a mass spectrometry; and the tandem mass spectrometry is
generally performed for the identification (qualitative analysis) of a
substance. On the other hand, in the quantitative analysis, a mass
spectrum is acquired without using the tandem mass spectrometry in many
cases. If an ion of a substance is given attention to, and subjected to a
qualitative analysis by the tandem mass spectrometry, data by the mass
spectrometry without using the tandem cannot be acquired during that
time, resulting in exhibiting a relation of substantially decreasing the
precision of the quantitative analysis. Hence, when a quantitative
analysis is performed, a control not to perform the tandem spectrometry
is needed. Therefore, conventionally, a qualitative analysis in which a
tandem mass spectrometry is first performed for the identification is
performed, and then, a quantitative analysis to acquire a mass spectrum
is performed.

[0004] According to the conventional procedure of the quantitative
analysis, a standard molecule is first analyzed in some concentrations.
Then, changes over time of the ion intensity (mass chromatogram) with
respect to m/z (mass/charge ratio) of ions originated from the standard
molecule are acquired to determine peak areas of the mass chromatograms.
A calibration curve is fabricated from the relation of the peak areas and
the concentrations of the sample substance. Next, the same substance
having an unknown concentration is analyzed to determine peak areas of a
mass chromatogram. The substance concentration corresponding to the peak
areas of the mass chromatogram is determined based on the fabricated
calibration curve. Since this method has a precondition that a standard
molecule is procured in advance to fabricate a calibration curve, the
application to unknown components as objects of marker searches is
difficult.

[0005] Patent Document 1 describes a method of performing a comparative
quantitation on unknown components whose calibration curves cannot be
fabricated, to search markers. This method involves first acquiring data
for the analysis on a standard sample containing various components, then
acquiring data for the analysis on another sample expected to contain the
same components, and calculating the ion intensity ratio (or the peak
area ratio) for each component. Then, standard ion intensity ratio is
determined, and the ion intensity ratio for the each component is
normalized using the value of the standard ion intensity ratio. Thereby,
the comparative ion intensity ratio for the each component can be
determined, and a component (marker candidate) exhibiting an outstanding
variation in the ion intensities can be specified. The identification of
a marker candidate separately needs an analysis of preferentially
performing a tandem mass spectrometry.

[0006] For unknown components whose calibration curves cannot be
fabricated, the comparative quantitation can be performed by using the
isotope labeling technique. That is, a comparative quantitative analysis
can be performed by mixing a sample containing various components and an
isotope-labeled standard sample (Non-patent Document 1). An
isotope-labeled component is simultaneously detected with an unlabeled
component, but since values of m/z of detected ions are different by a
predetermined value, comparing the ion intensities (or peak areas) in a
mass chromatogram of the ion pair enables determination of the
concentration ratio of the each component. Employment of this means
enables performance of the quantitation with no problem. However, this
means not only has a limitation on the types of samples applicable, but
also requires much time and costs. Hence, in marker searches, the isotope
labeling technique is not employed in many cases.

[0007] For an interface of an LC/MS, spray ionization methods such as the
electrospray ionization method (ESI), the atmospheric pressure chemical
ionization method (APCI), the atmospheric pressure photoionization method
(APPI), and the like are used. In an interface using a spray ionization
method such as ESI, the pneumatically assisted ESI or the sonic spray
ionization method (SSI), quantitative analysis-inhibitory factors named
as the matrix effect, the ion suppression and the ion enhancement are
known to occur. The matrix effect and the ion suppression are phenomena
in which scrambling for charges between components as described below
cause variations in ion intensities. Even in the case of performing a
comparative quantitative analysis as described in Patent Document 1, if
quantitative analysis-inhibitory factors such as the matrix effect and
the ion suppression occur, analysis results of data for the analysis may
lose the reliability. In this connection, the matrix effect is a
phenomenon in which in the case where a sample contains a large amount of
ionic components, the ion intensity is reduced, and the matrix effect can
be avoided if desalting is sufficiently carried out in preparation of a
sample.

[0008] The ion suppression occurs in the case where the amount of object
components to be ionized is equal to or more than the maximum value of
the ion amount which can be generated in the interface (ionization unit).
If this phenomenon occurs, scrambling for charges between various
components occurs, and the ionization efficiency is decreased depending
on chemical properties and amounts of each component. As a result, the
relation between the ion intensities to be detected and component
concentrations loses linearity (Non-patent Document 2). The maximum value
I of ion amounts which can be generated has, if Q represents a liquid
flow rate; κ represents an electric conductivity of a liquid; and
γ represents a surface tension, the following relation:

I=β(ε)(Qκγ/ε)1/2 (1)

wherein β is a constant; and ε is a dielectric constant of
the liquid. In order to beforehand prevent the ion suppression from
occurring, it is indicated from the expression (1) that raising the
electric conductivity κ of a liquid high is effective. However, too
high an electric conductivity κ decreases the ion generation
efficiency. Hence, it is desirable that κ be set in the range of
being capable of generating ions efficiently by addition of an acid and
the like to a mobile phase. In other words, since the electric
conductivity κ needs to satisfy two contradictory necessities, the
electric conductivity κ cannot actually be made high enough to
reduce the ion suppression. Therefore, it is difficult to effectively
prevent the ion suppression phenomenon regardless of conditions.

[0009] The problem as described above may occur not only in the spray
ionization method such as ESI but also in other interfaces in LC/MS such
as the atmospheric pressure chemical ionization method (APCI) and in
ionization units of GC/MS. This is because the maximum ion amount which
can be generated in an interface (ionization unit) has an upper limit.

[0010] On the other hand, the ion enhancement is caused by an increase in
the maximum ion amount which can be generated in an interface (ionization
unit) due to an increase in ionic components contained in a sample. As a
result, the ionization efficiency is increased depending on chemical
properties and amounts of each component, and the ion intensity to be
detected increases, which is a phenomenon of the ion enhancement.

[0011] Methods for detecting the occurrence of quantitative
analysis-inhibitory factors such as the ion suppression include
monitoring of the ion intensity using an internal standard. For example,
Non-patent Document 3 describes an evaluation method of the sample
preparation by using an isocratic LC, (flow rate: 0.25 mL/min), whose
mobile phase component is constant, and introducing an internal standard
by infusion (flow rate: 5 μL/min) from the downstream side of a
separation column. In the case where a sample is not sufficiently
purified, quantitative analysis-inhibitory factors such as the ion
suppression occur due to influences by salts and the like contained in
the sample right after the introduction of the sample, and the intensity
of ions originated from the internal standard decreases. Monitoring this
ion intensity enables detection of quantitative analysis-inhibitory
factors such as the ion suppression and the matrix effect. However, since
this method ionizes a sample after the sample has been passed through a
relatively long passage after the LC separation, in the case where the LC
flow rate is small, the method is liable to give a decreased separation
precision; and the method is effective for a semi-micro LC, a
general-purpose LC and the like, whose LC flow rate is high, but the
method is difficult to apply to separation means such as a micro LC and a
nano LC (capillary LC), whose LC flow rate is low. Additionally, no
internal standard has been optimized.

[0012] In order to suppress the occurrence of quantitative
analysis-inhibitory factors, it is necessary that the sample preparation
be modified to raise the purity of a sample to remove impurities and the
like, which become quantitative analysis-inhibitory factors in the
sample, or the separation condition in LC be modified and the separation
be carried out or otherwise spending a more time to reduce the types of
various components contained in the separated components.

[0017] As described hitherto, in marker searches, it is required that
samples originated from disease subjects and healthy subjects be compared
to extract variant components; unknown component substances composed of a
large number of types constituting the variant components be identified
with high precision; and the quantitative analysis of the component
substances be performed with high sensitivity/high precision without
being influenced by quantitative analysis-inhibitory factors such as the
ion suppression phenomenon. Moreover, it is required that the series of
analyses be performed in as short a time as possible, that is, a high
throughput is required. It is also needed that the series of analyses be
performed in as low a cost as possible.

[0018] For the requirements, since the analysis using a calibration curve,
which is conventional means for the quantitative analysis, needs to
acquire calibration curve data from a standard molecule in advance, the
analysis cannot be applied to quantitative analyses containing unknown
substances. The analysis using the isotope labeling technique as
described in Non-patent Document 1 also limits the types of samples, and
requires much time and a high cost. The comparative quantitation means
described in Patent Document 1 cannot avoid an influence of the variation
in the ion intensity due to quantitative analysis-inhibitory factors, so
there arises a problem that the precision of the analysis result
decreases.

[0019] Then, performing analyses not using the isotope labeling technique
and not being influenced by quantitative analysis-inhibitory factors such
as the ion suppression is an object for marker searches. However, in the
case of performing the quantitation without using the isotope labeling
technique, if quantitative analysis-inhibitory factors occur, there
arises a need for reperforming the analysis. Hence, making the number of
times of the qualitative analysis and that of the quantitative analysis
as few as possible is important from the viewpoint of cost and speed.

[0020] For achieving a high throughput, there are problems as follows.
Marker searches need a quantitative analysis to acquire a mass spectrum
without performing the tandem mass spectrometry and a qualitative
analysis to perform a tandem mass spectrometry for the identification.
Additionally, samples composed of very many components are analyzed in
many cases, and in this case, it is difficult in many cases to perform
the qualitative analysis for all the components detected by one time of
the analysis (JP Patent Publication (Kokai) No. 2005-091344A). This is
because the throughput of the tandem mass spectrometry is limited.
Therefore, there arises a need for many times of tandem mass
spectrometry, and the throughput of a series of marker searches cannot be
raised.

[0021] As detection means of quantitative analysis-inhibitory factors,
since means described in Non-patent Document 3 cannot secure a precision
in the quantitative analysis using LC having a low liquid flow rate and
gradient mode LC, there is a problem that the means cannot perform
high-sensitive analyses by LC having a low liquid flow rate, and analyses
of trace amounts of samples.

[0022] In the case of a low LC flow rate, an internal standard needs to be
injected from the upstream of a separation column, but therefor, the
internal standard needs not to be adsorbed on the separation column. This
need is a very important problem not only in an isocratic mode, in which
the mixing ratio of organic solvents in an LC mobile phase is constant,
but also in a gradient mode, in which the ratio changes in terms of time.
Hence, the chemical properties of the internal standard need to be fully
considered.

[0023] Further, in order to detect the occurrence of quantitative
analysis-inhibitory factors such as the ion suppression, the mixing ratio
of organic solvents in an LC mobile phase needs to be considered from the
viewpoint of chemical properties of an internal standard. That is, the
case of an aqueous mobile phase having a very low organic solvent ratio,
and the case of a mobile phase having a very high organic solvent ratio
are believed to be different in optimum chemical properties of the
internal standard.

[0024] An object of the present invention is to solve the above-mentioned
problems, and to perform the qualitative analysis and quantitation not
using the isotope labeling technique and not being influenced by
quantitative analysis-inhibitory factors and with high sensitivity and
high throughput. The object of the present invention is to provide an
analysis method for acquiring reliable data in a smallest number of times
of qualitative and quantitative analyses without using the isotope
labeling technique.

[0025] Another object of the present invention is to provide an internal
standard to detect the occurrence of quantitative analysis-inhibitory
factors.

[0026] Further another object of the present invention is to provide an
automatic analyzer and an automatic diagnosing apparatus using an
internal standard to detect the occurrence of quantitative
analysis-inhibitory factors. This is because it is believed to be
necessary to detect the occurrence of quantitative analysis-inhibitory
factors in the case where a chemical analog expected to have chemical
properties similar to an object substance for the analysis is
quantitatively analyzed as a standard reagent.

Means for Solving the Problems

[0027] In order to solve the above-mentioned problems, means for the
analysis described below is provided. That is, first, an internal
standard to be detected simultaneously with a component for the analysis
is mixed in a mobile phase or an eluted liquid of a liquid chromatograph;
and a mass chromatogram of ions originated from the internal standard is
acquired under the condition where no quantitative analysis-inhibitory
factors occur, and recorded in a data analysis unit. Typically, the blank
sample containing no sample for the analysis is analyzed. Then, a sample
for the analysis is mixed; data for the analysis of the sample are
acquired; and at this time, the intensity of ions originated from the
internal standard is compared with that in the analysis of the blank
sample in an analysis real time in the data analysis unit. At this time,
if an inconsistency is detected between the ion intensities, quantitative
analysis-inhibitory factors are determined to have occurred in mixing of
the sample for the analysis; and in this case, since the precision of the
qualitative analysis result decreases due to the quantitative
analysis-inhibitory factors, the analysis mode is changed from the
quantitative analysis mode taking a low preference to the tandem mass
spectrometry to the qualitative analysis mode taking preference to the
tandem mass spectrometry. Then if the intensity of ions originated from
an internal standard becomes consistent with that of a blank sample in
the analysis real time by decreasing the mixing amount of a sample for
the analysis, or otherwise, the analysis mode is again changed to the
quantitative analysis mode. In the case where there arises a time region
in a mass chromatogram where the intensities of ions originated from an
internal standard are consistent, data for the analysis of the sample in
the time region of the consistency are acquired as effective data for the
analysis. In this means for the analysis, an internal standard to be used
is a substance having properties stably detected during the analysis real
time.

[0028] As a substance sensitively reacting to quantitative
analysis-inhibitory factors such as the ion suppression, an internal
standard described below is provided. That is, in the analysis of
positive ion, and in the case where a mobile phase is an aqueous one
having a low organic solvent ratio therein, a substance is provided which
has an isoelectric point or an (acid) dissociation constant not
remarkably lower than pH (hydrogen ion concentration) of the mobile
phase, and has a high hydrophilicity. Then in the case of a high organic
solvent ratio, a substance is provided which has an isoelectric point or
a dissociation constant not remarkably lower than pH of the mobile phase,
and has a hydrophobicity. On the other hand, in the analysis of negative
ion, and in the case where a mobile phase is an aqueous one having a low
organic solvent ratio therein, a substance having a basicity and a high
hydrophilicity is provided. That is, a substance is provided which has an
isoelectric point or dissociation constant of higher than 8, and a low
hydrophobicity. In the case of a high organic solvent ratio, a substance
having a high basicity and a hydrophobicity is provided.

[0029] In the case of using a liquid chromatograph in a gradient mode in
which the organic solvent ratio in a mobile phase varies in terms of
time, an internal standard having a high hydrophilicity and an internal
standard having a hydrophobicity are concurrently used according to the
variation range of the organic solvent ratio. By properly using the ion
intensity information of ions originated from the internal standards
according to the organic solvent ratio in a mobile phase, and reflecting
the information on the analysis result, a quantitative analysis with high
precision can be performed.

[0030] In the case of switching positive and negative ion detection modes
at a high speed in one time of LC/MS analysis for an analysis, both of
internal standards for the analysis for positive ion and the analysis for
negative ion having different isoelectric points or dissociation
constants from each other are mixed in a mobile phase or an eluate, and
data are then acquired. By properly using the ion intensity information
of ions originated from the internal standards based on positive and
negative ion mode, and reflecting the information on the analysis result,
a quantitative analysis with high precision can be performed.

[0031] In order to further improve the efficiency, means is also provided
in which two types of internal standards exhibiting largely different
sensitivities to analysis-inhibitory factors are introduced; a sample for
the analysis is mixed therein, and analyzed; and by comparing mass
chromatograms of ions originated from both the internal standards, the
occurrence of analysis-inhibitory factors is detected by one time of the
analysis. As two types of internal standards having largely different
sensitivities to analysis-inhibitory factors, two types of internal
standards having different isoelectric points are selected.

[0032] Further, means is also provided in which one type of an internal
standard is introduced; additionally a substance present in an analysis
solution and capable of becoming a second internal standard is searched
for to make a second internal standard; and by comparing mass
chromatograms of the both, the occurrence of analysis-inhibitory factors
is detected by one time of the analysis.

[0033] As an analyzer to solve the above-mentioned problems, an analyzer
is further provided which has a mobile-phase introduction unit to mix an
internal standard and introduce a mobile phase, and a sample introduction
unit, a separation unit, an ionization/mass-analysis unit, a data
analysis unit, and a display unit, and has means to acquire and save a
first mass chromatogram of ions originated from an internal standard in
the state of not being mixed with a sample for the analysis, and means to
acquire and compare a second mass chromatogram of ions originated from
the internal standard in the state of being mixed with the sample for the
analysis, and means to collect data for the analysis in the case where
the inconsistency between the first and the second mass chromatograms is
a given value or less.

[0034] An apparatus is also provided which has means to acquire and
compare mass chromatograms of a first and a second internal standards in
the state that the both are mixed in a mobile phase, and to collect data
for the analysis according to the comparison result. An apparatus also is
further disclosed which, in the state that a first internal standard is
mixed in a mobile phase, has means to monitor data for the analysis to
search for another substance capable of becoming a second internal
standard in an analysis solution, and acquires and compares mass
chromatograms of the second internal standard obtained by the search and
the first internal standard, and collects data for the analysis according
to the analysis result.

[0035] An apparatus also is further disclosed in which by mixing an
internal standard in a sample for the analysis, and performing an
analysis of the mixture, the presence/absence of the occurrence of
analysis-inhibitory factors is detected, and in the case where the
factors are significantly detected, a protocol for preparing a sample for
the analysis is partially altered and a reanalysis is performed.

[0036] The present description includes the subject described in the
specification and/or the drawings of Japanese Patent Application
2008-096710, which is the basis of the priority to the present
application.

Advantages of the Invention

[0037] An internal standard is mixed in a mobile phase or an eluate of LC,
and the blank sample is first analyzed under the condition where no
analysis-inhibitory factors occur. Next, a sample for the analysis is
analyzed, but at this time, by measuring the intensity of ions originated
from the internal standard in the analysis real time, and comparing with
an analysis result of the blank sample in the analysis real time, whether
or not the analysis-inhibitory factors have occurred when the sample for
the analysis has been mixed can be detected with high precision. Further,
based on the inconsistency above, the error in quantitative data can be
evaluated.

[0038] Data in a time region indicating a consistency by comparison of
mass chromatograms of ions originated from internal standards of the
blank sample and the mixed sample for the analysis are judged not to be
influenced by analysis-inhibitory factors, and data for the analysis of
the sample for the analysis in the time region where the ion intensities
are consistent with each other are acquired as effective data for the
analysis, thereby enabling elimination of wasteful analysis time, and
improve the analysis efficiency.

[0039] By introducing two types of internal standards, and acquiring and
comparing mass chromatograms by one time of the analysis, the occurrence
of analysis-inhibitory factors can be detected within the real time of
one time of the analysis, thereby enabling further reduction in the
analysis time.

[0040] By mixing an internal standard in a mobile phase of LC in advance,
micro LC and nano LC (capillary LC), whose liquid flow rate is low, can
be used for the quantitative mass spectrometry. This exhibits an
advantage to a high-sensitive analysis, and enables analysis of a trace
amount of a sample.

[0041] In the case where quantitative analysis-inhibitory factors occur,
since very many components are detected simultaneously in many cases, by
preferentially performing a qualitative analysis, the total number of
times of analyses can be reduced, enabling achievement of a high
throughput.

[0042] The identification of a component for the detection needs to hold
the mass precision of a mass spectrometer high. However, in data
acquisition using LC, variations in m/z values are detected in the
analysis in some cases. Then, by always monitoring m/z values of ions
originated from an internal standard (known substance) detected, the m/z
value of ions for the detection can be corrected to a right m/z value.
Thereby, data for the analysis having an extremely high mass precision
can be acquired.

[0043] In the case where no quantitative analysis-inhibitory factors can
be confirmed to occur, if the ion intensity (mass chromatogram area) of
ions for the detection is normalized based on the intensity of ions
originated from an internal standard, the comparison between data can be
carried out with high precision. This is because the ion intensity varies
more or less for each analysis in some cases.

[0044] In a mass chromatogram corresponding to an internal standard, and
in the case where the ion intensity decreases below a predetermined
inconsistency (threshold), the occurrence of quantitative
analysis-inhibitory factors such as the ion suppression is detected.
Since the decreasing rate gives the upper limit of the decreasing rate of
the intensities of other ions detected during the time, the decreasing
rate can be reflected to errors in intensities or areas of the other
ions. By contrast, in a mass chromatogram corresponding to an internal
standard, and in the case where the ion intensity increases above a
predetermined inconsistency (threshold), the occurrence of quantitative
analysis-inhibitory factors such as the ion enhancement is detected.
Since the increasing rate gives the lower limit of the increasing rate of
the intensities of other ions detected during the time, the increasing
rate can be reflected to errors in intensities or areas of the other
ions.

[0045] Further in an automatic analyzer and a diagnosing apparatus, and in
the case where quantitative analysis-inhibitory factors such as the ion
suppression are detected, by partially altering a method for preparing a
sample for the analysis, and performing a reanalysis, a quantitative
analysis can be performed under the condition where no quantitative
analysis-inhibitory factors are detected.

[0046] Additionally, in an automatic analyzer and a diagnosing apparatus,
by using the information of the ion intensity of ions originated from an
internal standard and the ion intensity of ions originated from a
standard molecule, a presumed (corrected) value of quantitative data and
an error thereof can be reflected to output data.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 is a constitution diagram of an Example in a mass
spectrometry system according to the present invention.

[0048]FIG. 2 is a mass chromatogram of ions originated from an internal
standard in typical blank-sample data for the analysis.

[0049]FIG. 3 is a diagram showing a comparison of mass chromatograms of
ions originated from internal standards in data for the analysis of a
blank sample and a sample for the analysis, and an example of detection
of the occurrence of quantitative analysis-inhibitory factors, in an
Example in a mass spectrometry system according to the present invention.

[0050]FIG. 4 is a diagram showing a comparison of mass chromatograms of
ions originated from internal standards in data for the analysis of a
blank sample and a sample for the analysis, and an example of a
large-scale occurrence of quantitative analysis-inhibitory factors.

[0051] FIG. 5 is a diagram showing a comparison of mass chromatograms of
ions originated from two types of internal standards in data for the
analysis of a sample for the analysis, and an example of detection of the
occurrence of quantitative analysis-inhibitory factors, in an Example, in
which the two types of internal standards were used, in a mass
spectrometry system according to the present invention.

[0052]FIG. 6 is an illustrative diagram of a screen of a data analysis
unit, or a screen of a control unit of a mass spectrometer in an Example
in a mass spectrometry system according to the present invention.

[0053] FIG. 7 is a constitution diagram of an Example in another mass
spectrometry system according to the present invention.

[0054]FIG. 8 is a diagram showing an example of the time dependency of
measured m/z of ions originated from an internal standard in a mass
spectrometry system according to the present invention.

[0055]FIG. 9 is a diagram interpreting analysis steps in First Example in
the present invention.

[0056] FIG. 10 is a diagram interpreting analysis steps in Second Example
in the present invention.

[0057]FIG. 11 is a diagram interpreting analysis steps in Third Example
in the present invention.

[0058]FIG. 12 is comparative diagrams of (a) a total ion chromatogram,
and (b) a mass chromatogram of ions originated from an internal standard,
acquired using a mass spectrometry system according to the present
invention.

[0059]FIG. 13 is comparative diagrams of mass spectra acquired in the
retention time (1) using a mass spectrometry system according to the
present invention.

[0060] FIG. 14 is comparative diagrams of mass spectra acquired in the
retention time (2) using a mass spectrometry system according to the
present invention.

[0061]FIG. 15 is a diagram showing the relation between the peak area and
the injection amount with respect to ions detected in the retention times
(1) and (2).

[0062]FIG. 16 is an illustrative plan view of an automatic analyzer
according to the present invention.

[0063] FIG. 17 is a sectional illustrative diagram of a turn table 301 and
a turn table 305 of an automatic analyzer according to an embodiment of
the present invention.

[0114] Hereinafter, embodiments according to the present invention will be
described by way of drawings.

EXAMPLE 1

[0115] FIG. 1 shows a constitution diagram of an Example in a mass
spectrometry system according to the present invention. The system
comprises a mobile-phase introduction unit 101, a sample introduction
unit 102, a separation unit 103, an ionization/mass-analysis unit 104, a
data analysis unit 105, a display unit 106, and a control unit of
analysis mode 107. The data analysis unit 105, the display unit 106 and
the control unit of analysis mode 107 are put together as a control
system 108. Each unit of the mobile-phase introduction unit 101, the
sample introduction unit 102, the separation unit 103, the
ionization/mass-analysis unit 104 and the control system 108 is
collectively controlled by a system control unit to supervise the whole
system, and desired operations are achieved while information of control
states is bilaterally exchanged between each unit of the system. A mobile
phase is introduced from the mobile-phase introduction unit 101; a sample
205 composed of various components is introduced through the sample
introduction unit 102; and the separation is carried out in the
separation unit 103 composed of separation devices such as a liquid
chromatograph (LC). A mobile phase A (201), a mobile phase B (202) as
well as a mobile phase C (203) are prepared to the mobile-phase
introduction unit 101. The mobile phases A and B are ones used in common
reverse-phase chromatograph, and a typical mobile phase A (201) is 2%
acetonitrile in water (0.1% formic acid); and a typical mobile phase B
(202) is 98% acetonitrile in water (0.1% formic acid).

[0116] The mobile phase C (203) is one in which a specified amount of an
internal standard 204 is added to the mobile phase A. In the case where,
from the start, internal standards are added to the mobile phases A and
B, the mobile phase C is unnecessary. The important point is that the
internal standard 204 is introduced to the separation unit 103 always in
a constant concentration. Samples (blank sample, sample for the analysis,
and the like) 205 introduced to the sample introduction unit 102 are
separated in the separation unit 103, introduced to the
ionization/mass-analysis unit 104 sequentially as separated components,
and ionized and mass analyzed. The output of the mass analysis unit is
introduced to the data analysis unit 105, and stored and data treated as
data for the analysis. The data analysis unit 105 shown in FIG. 1 is
provided with the display unit 106, which displays information indicating
the priority in the tandem mass spectrometry, including "quantitative
analysis-preferential mode" and "qualitative analysis-preferential mode"
in the analysis real time. Additionally, the display unit 106 displays
the time dependency of the total ion current (total ion current
chromatogram), and analysis situations such as the latest mass spectrum
or tandem mass spectrometry spectrum. The control of the mass-analysis
unit 104 may be carried out by the data analysis unit 105, or may be
carried out by a separate information processing facility (control unit
for the analysis mode 107) as shown by a dashed line. Alternatively, the
control may be carried out using a constitution in which the display unit
106 has a selection button for the analysis mode, as described later, and
an operator can switch the modes.

[0117] The outline of the analysis procedure is as follows. First, mobile
phases A, B and C as described before are prepared; while the ratio of
the mobile phase C containing an internal standard is held at a constant
value, for example, 3%, the mixing ratio of the mobile phase A and the
mobile phase B is set at an initial constant value, and is varied over
the time. Typically, the mixing ratio is linearly varied, for example,
such that the mixing ratio at the start of the mobile phases A and B is
set at 92% and 5% (C is fixed at 3%), and the ratio at the end after 60
min is set at 47% and 50%. The values of this mixing ratio at the start
and the end, the mixing time and the like are just an example, and can
suitably be changed. The gradient mode, in which the mixing ratio of the
mobile phases A and B is varied in terms of time, is means often used in
LC separation. In LC separation in the pharmacokinetic analysis requiring
a high throughput analysis, the isocratic mode, in which the mixing ratio
of the mobile phases A and B is fixed in terms of time, is often
utilized.

[0118] As an internal standard, a desired substance is selected and
prepared in advance according to the conditions described later. An
internal standard is selected so as to be always detected in an LC
retention time range where the internal standard is detected
simultaneously with a component for the analysis, and mixed in a mobile
phase or a component of a mobile phase; a blank sample is introduced from
the sample introduction unit 102; and a mass chromatogram of ions
originated from the internal standard is acquired, and recorded in the
data analysis unit 105. In the data analysis unit 105, the data storage
means 113 constituted of a data storage medium and the like is disposed.
At this time, data need to be acquired under the conditions where no
quantitative analysis-inhibitory factors occur. Therefore, the same
substance as a mobile phase A, or pure water is introduced as a blank
sample to be introduced through the sample introduction unit 102, and no
substance other than substances previously present in the mobile phase is
mixed. Caution needs to be taken so the blank sample as to contain no
impurities. In order for the internal standard itself to cause no ion
suppression, the internal standard needs to be contained in only a
minimum amount necessary for ion detection. Then, a sample for the
analysis is introduced through the sample introduction unit 102 in place
of the blank sample; and the data for the analysis of the sample mixed
with the same amount of the internal standard is acquired, and recorded
in the data analysis unit.

[0119] A typical mass chromatogram of ions originated from an internal
standard is shown in FIG. 2. The abscissa indicates the retention time of
LC; and the ordinate indicates the ion intensity. The data is acquired
using a liquid chromatograph/mass spectrometer (LC/MS). Since LC in LC/MS
usually uses a reverse-phase column as a separation column, it is
desirable that the internal standard have a high hydrophilicity in such a
degree that the internal standard can pass through the reverse-phase
column without adsorption. If the hydrophilicity is very high, since ions
originated from the internal standard contained in the mobile phase are
always stably detected in the retention time of the separation, the
occurrence of quantitative analysis-inhibitory factors can always be
monitored. In the example of FIG. 2, as the internal standard, a
synthetic peptide whose amino acid sequence is SSSSSSK was used. As a
blank sample, pure water was used. In the present Example, when the blank
sample of pure water was introduced at a timing of the retention time of
0, pure water was eluted after 12.6 min. Hence, the ion intensity 1201 of
ions originated from the internal standard in FIG. 2 drops at a retention
time of 12.6 min, and ions originated from the internal standard come not
to be detected. In this time range, components contained in the sample
and not having being separated are eluted, but if the components are
considered to be out of the object of the quantitation, even if the
occurrence of quantitative analysis-inhibitory factors cannot be
monitored, there arises no problem. By contrast, in the other time range,
the ion intensity exhibits a very low dependency of the ion intensity on
the retention time, and changes only smoothly. This can interpret that
the ionization efficiency of the internal standard does not change
outstandingly due to the change in the components of the mobile phase.

[0120] The principle of the ion suppression being a quantitative
analysis-inhibitory factor will be described simply hereinafter. In the
spray ionization method such as the electrospray ionization method,
first, charged liquid droplets of nearly micron size are produced by
spraying of the liquid. In the charged liquid droplets, ions of a liquid
phase distribute on the liquid droplet surface by the electrostatic
repulsive force. Then, solvent molecules evaporate from the charged
liquid droplets, and gaseous ions are produced from the charged liquid
droplets through the ion evaporation process or the charge residue
process. Hence, the major origin of the gaseous ions to be detected by a
mass spectrometer is ions of a liquid phase present on the surface of the
charged liquid droplets. The size of the charged liquid droplets
decreases due to the evaporation of the solvent molecules, and the charge
density of the surface increases. Thereby, also in a non-charged object
substance for the analysis in the vicinity of the liquid droplet surface,
the ionization (addition of protons, and the like) progresses. In the
case where the amount of the object substance for the analysis is
sufficiently small with respect to the charge of the liquid droplet
surface, the ionization efficiency of the object substance for the
analysis becomes constant. In the case where this condition is satisfied,
the relation between the intensity of ions for the detection and the
sample amount becomes constant. However, in the case where the amount of
the object substances for the analysis is equal to or more than the
charge of the liquid droplet surface, the supply of the charge to the
object substance for the analysis becomes partially insufficient, and
scrambling of the charge between the object substances for the analysis
occurs on the liquid droplet surface. As a result, the efficiency of
production of gaseous ions from the charged liquid droplet varies
(decreases). That is, this is the occurrence of the ion suppression. The
decreasing rate of the ionization efficiency depends on physicochemical
properties of the object substance for the analysis. According to the ion
production process described above, the main factors characterizing an
object substance for the analysis susceptible to the influence of the ion
suppression are considered to be two points of 1) the lowness of the
degree of electrolytic dissociation in a liquid phase (or a property of
charged liquid droplets charging to the reverse polarity), and 2) the
easiness of access to the liquid droplet surface (the lowness of the
surface activity). The lowness of the surface activity can be expressed
in hydrophobicity and hydrophilicity. By contrast, components which
electrolytically dissociate completely in a liquid phase can be present
on the charged liquid droplet surface as ions, and are considered to be
hardly susceptible to the influence of the ion suppression. On the other
hand, the quantitative analysis-inhibitory factor such as the ion
enhancement occurs due to a rapid increase of ionic substances. In this
case, as a result of an increased charge of the liquid droplet surface,
the ionization efficiency of an object substance for the analysis
increases. The increasing rate of the ionization efficiency also depends
on physicochemical properties of the object substance for the analysis,
and factors characterizing the influence are the same as the case of the
ion suppression.

[0121] In selection of an internal standard to monitor the occurrence of
quantitative analysis-inhibitory factors, the present inventors have
found for the first time that an isoelectric point or a dissociation
constant (or acidity/basicity), and hydrophobicity/hydrophilicity of an
internal standard can be used as indices. The isoelectric point refers to
a pH at the time when an ampholyte compound exhibits an average charge of
0 as the whole compound. An internal standard having an isoelectric point
lower than 7 is acidic, and was confirmed to be advantageous to the
detection of quantitative analysis-inhibitory factors because it reacts
sensitively to the quantitative analysis-inhibitory factors in the
analysis for positive ion. Generally, an acidic molecule having a
dissociation constant (pK) lower than 7 reacts sensitively to
quantitative analysis-inhibitory factors in the analysis for positive
ion, which is advantageous to the detection of the quantitative
analysis-inhibitory factors. By contrast, a basic molecule having a
dissociation constant (pK) or an isoelectric point higher than 7 reacts
sensitively to quantitative analysis-inhibitory factors in the analysis
for negative ion, which is advantageous to the detection of the
quantitative analysis-inhibitory factors. On the other hand, with respect
to the hydrophobicity (or hydrophilicity), in a mobile phase having a low
ratio of an organic solvent, it is a necessary condition that an internal
standard has a lower hydrophobicity (or higher hydrophilicity) than G or
A being an amino acid, which has an average hydrophobicity (or
hydrophilicity). In a mobile phase having a ratio of an organic solvent
of more than 50%, it is a necessary condition that an internal standard
has a higher hydrophobicity than G or A. In the case of a mobile phase
used in the reverse-phase LC/MS, an acid such as formic acid is added to
the mobile phase to adjust pH thereof to about 3 in many cases from the
viewpoint of a balance between LC separability and ionization. Therefore,
in the case where the isoelectric point is sufficiently lower than pH of
a mobile phase, there arise a possibility that the production efficiency
of positive ions becomes too low, which may be to be taken into account.

[0122] In the case of too low an ionization efficiency, an internal
standard needs to be mixed in a mobile phase in a high concentration, so
the internal standard is nor preferable. This is because, since the
occurrence of quantitative analysis-inhibitory factors depends on the
amount of substances to be ionized, the addition itself of the internal
standard can possibly cause quantitative analysis-inhibitory factors.
From the viewpoints of the above, in the case of analyzing positive ions
using a mobile phase having a low ratio of an organic solvent, synthetic
peptides (acidic peptides such as DSSSSS and EQQQQQ, the isoelectric
points are 3.8 and 4.0, respectively) having a high hydrophilicity and an
isoelectric point of 3 or higher and 8 or lower are most suitable as an
internal standard. Even compounds (not peptides) having a dissociation
constant of 7 or more can be of course used as an internal standard. The
dissociation constant (pK) or the isoelectric point is most suitably 4 or
lower. On the other hand, basic peptides (SSSSSK and SSKSSK, the
isoelectric points are 8.5 and 10.0, respectively) having an isoelectric
point of 8 or higher, basic compounds having a dissociation constant of 8
or higher, and the like can be similarly used as an internal standard.
Here, basic compounds are less influenced by quantitative
analysis-inhibitory factors than acidic compounds, which is
disadvantageous to the detection, and additionally have a tendency of
easily producing not only singly-protonated molecules but also
multiply-protonated molecules. Polyprotonated molecules (polyvalent ions)
may be subjected to deprotonation by the gas-phase ion-molecule reaction
to become monovalent ions. This fact corresponds to the decrease of the
ion intensity, and means that it may become difficult to distinguish from
the detection of quantitative analysis-inhibitory factors.

[0123] Therefore, in the case of a mobile phase having a low ratio of an
organic solvent, basic compounds having an isoelectric point of 8 or
higher, which easily produce polyvalent ions, are unsuitable for the
detection of quantitative analysis-inhibitory factors. Summarizing the
above, in the case where positive ions are analyzed using a mobile phase
having a low ratio of an organic solvent, substances most suitable for an
internal standard are ones, which have a low hydrophobicity (a high
hydrophilicity), and are acidic and have a low value of an isoelectric
point or a dissociation constant in the range of 4 or lower and 2 or
higher, and in which only a singly-protonated molecule is detected, that
is, substances having an isoelectric point or a dissociation constant of
about 2 to 8. Taking peptides as an example, amino acids having an
isoelectric point of 3 or lower are D (isoelectric point: 2.8) only, and
most of the components have an isoelectric point of 3 or higher. It is
therefore conceivable that if an internal standard having a property of
the isoelectric point or dissociation constant of about 3 is used, ions
originated from the internal standard are most strongly influenced by
quantitative analysis-inhibitory factors such as the ion suppression. It
suffices if, as peptides utilizable as such an internal standard,
peptides containing D and E, which are typical acidic amino acids, and S,
Q, N and the like, which have a high hydrophilicity and hardly
electrolytically dissociate, in the amino acid sequence are selected; and
DSSSSS, ENNNNN and the like are suitable. (D and E have a high
hydrophilicity, and a pKR (side chain) of about 4.) On the other
hand, in the case of analyzing negative ions, substances most suitable as
an internal standard are ones having a high hydrophilicity (low
hydrophobicity), and additionally a basicity and a high isoelectric
point. It suffices if, as peptides utilizable as such an internal
standard, peptides containing R and K, which are typical basic amino
acids, and S, Q, N and the like, which have a high hydrophilicity, in the
amino acid sequence are selected. R and K also have a high
hydrophilicity, and additionally an isoelectric point of 9 or higher, and
a pKR (side chain) of 10 or higher. By contrast, S, Q and N not only
have a high hydrophilicity, but also have a property of hardly
electrolytically dissociating in a liquid phase. Examples of such
peptides include KNNNNN and RNNNNN, whose isoelectric points are 8.75 and
9.75, which are 8 or higher, respectively. Of course, there is no reason
that an internal standard must be a peptide, and any compound having the
above-mentioned properties can be used similarly.

[0124] In the case of analyzing positive ions using a mobile phase having
as high a ratio of an organic solvent as exceeding 50%, synthetic
peptides having a hydrophobicity, and an isoelectric point of 8 or lower
can be used as an internal standard. Particularly peptides and other
compounds having an isoelectric point or a dissociation constant of 4 or
lower are most suitable as an internal standard. On the other hand, basic
peptides having an isoelectric point of 8 or higher, basic compounds
having a dissociation constant of 7 or higher, and the like can similarly
be used as an internal standard. However, basic compounds have smaller
influence by quantitative analysis-inhibitory factors than acidic
compounds, which is disadvantageous to the detection, and additionally
have a tendency of easily producing not only singly-protonated molecules
but also multiply-protonated molecules. Polyvalently protonated molecules
(polyvalent ions) may be subjected to deprotonation by the gas-phase
ion-molecule reaction, and converted to monovalent ions. This fact
corresponds to the decrease of the ion intensity, and means that it may
become difficult to distinguish from the detection of quantitative
analysis-inhibitory factors.

[0125] Therefore, in the case of a mobile phase having a high ratio of an
organic solvent, basic compounds having an isoelectric point of 8 or
higher, which easily produces multiply-charged ions, are unsuitable for
the detection of quantitative analysis-inhibitory factors. Summarizing
the above, in the case of analyzing positive ions using a mobile phase
having a high ratio of an organic solvent, substances most suitable as an
internal standard are ones which have a hydrophobicity, and are
additionally acidic and have low values of the isoelectric point or
dissociation constant, and in which only singly-protonated molecules can
be detected, that is, substances having an isoelectric point or a
dissociation constant in the range of 2 to 8. Taking peptides as an
example, amino acids having an isoelectric point of 3 or lower are D (the
isoelectric point: 2.8) only, and most of components have an isoelectric
point of 3 or higher. It is therefore conceivable that if an internal
standard having a property of an isoelectric point or dissociation
constant of about 3 is used, ions originated from the internal standard
are most strongly influenced by quantitative analysis-inhibitory factors
such as the ion suppression. It suffices if, as peptides utilizable as
such an internal standard, peptides containing D and E, which are typical
acidic amino acids, and G, F, L and the like, which have a hydrophobicity
and are hardly electrolytically dissociated, in the amino acid sequence
are selected, and acidic peptides such as FDFGF and EFGFGF (the
isoelectric points are 3.8 and 4.0, respectively) are suitable. (D and E
have a high hydrophilicity, and additionally have a pKR (side chain)
of about 4.) On the other hand, in the case of analyzing negative ions,
substances most suitable as an internal standard are ones which have a
hydrophobicity, and are basic and have an isoelectric point or a
dissociation constant of 8 or higher. It suffices if, as peptides
utilizable as such an internal standard, peptides containing R and K,
which are typical basic amino acids, and G, F, and the like, which have a
hydrophobicity, in the amino acid sequence are selected. R and K have a
high hydrophilicity, and an isoelectric point of 9 or higher, and a
pKR (side chain) of 10 or higher. On the other hand, G, F and L not
only have a hydrophobicity, but also have a property of hardly
electrolytically dissociating in a liquid phase. Examples of such
peptides include KGGGGG and RFFFFF, whose isoelectric points are 8.75 and
9.75, respectively, which are 8 or higher. Of course, there is no reason
that an internal standard must be a peptide, and any compound having the
above-mentioned properties can be used similarly.

[0126] In the case of using a liquid chromatograph in a gradient mode, the
ratio of an organic solvent in a mobile phase varies in terms of time.
Nevertheless, in the case where the ratio of an organic solvent is always
50% or less, an internal standard having a low hydrophobicity can be
used. In the case where the ratio of an organic solvent is always 70% or
more, an internal standard having a hydrophobicity can be used. However,
in the case where the ratio of an organic solvent varies from 40% to 90%,
use of only one of an internal standard having a low hydrophobicity and
an internal standard having a hydrophobicity is not preferable to perform
a quantitative analysis with high precision. In this case, concurrent use
of the both is preferable. According to the ratio of the organic solvent
in the mobile phase, the presence/absence of, and the degree (error) of
the influence of the occurrence of quantitative analysis-inhibitory
factors based on information of the ion intensities of ions originated
from each of the internal standards are reflected to the analysis
results, thereby enabling performance of a quantitative analysis with
high precision. In this case, if the occurrence of quantitative
analysis-inhibitory factors is detected based on information of the ion
intensity of ions originated from one of the internal standards, even if
the occurrence is not detected based on that of the other internal
standard, the occurrence of quantitative analysis-inhibitory factors is
confirmed. In the case where the occurrence of quantitative
analysis-inhibitory factors is detected by ions originated from both the
internal standards, the one having a larger variation can be reflected to
the quantitative error. In the case of using an analysis mode in which
positive and negative ion detection modes are switched at a high speed in
one time of LC/MS analysis, data for the analysis of positive and
negative ions can be acquired simultaneously. In this case, both of
internal standards for the analysis for positive ion and for the analysis
for negative ion are mixed in an LC mobile phase or an eluate to acquire
data, and the information of the ion intensities of ions originated from
the internal standards are separately used based on positive and negative
ion mode, and reflected to the analysis results, thereby enabling a
quantitative analysis with high precision.

[0127] Finally, the molecular weight of an internal standard needs to be
examined. If m/z of ions originated from an internal standard overlaps on
m/z of other ions for the detection to such a degree that the overlap
cannot be distinguished by the mass resolving power of a mass
spectrometer, there arises a possibility of false recognition in which
the ion intensity originated from the internal standard has increased. In
this case, as described later, the detection of the ion suppression
becomes difficult. For example, in the analysis of peptides, if m/z of
ions originated from an internal standard is 600 or more or 350 or less,
it can be expected from experience that a possibility that the m/z
overlaps on m/z of other ions for the detection is very low. In the case
of analyzing a low-molecular compound, the compound of m/z of 500 or more
is rare, and if m/z of ions originated from an internal standard is 400
or more, no problem is conceivable. Generally, since an internal standard
having a higher molecular weight has a tendency of exhibiting a higher
hydrophobicity, the molecular weight is desirably 1,000 or lower.

[0128] In the analysis using such an internal standard, the occurrence of
quantitative analysis-inhibitory factors decreases or increases the ion
intensity originated from the internal standard. At the same time, the
ion intensities of other ions also may possibly decrease or increase.
When the ion suppression occurs, the upper limit of the decreasing rate
of the ion intensity becomes a deceasing rate of the ion intensity of
ions originated from the internal standard. By utilizing this property,
the decreasing rate of the ion intensity of ions originated from an
internal standard can be included in the error as a maximum decreasing
rate in a measurement value of the ion intensity of other components, and
can be reflected to statistical processes of various data. Since
actually, data for the analysis always contain a measurement error, it
suffices that a threshold is set based on the error; the threshold is
employed as an error in the case where the decreasing rate of the ion
intensity of ions originated from an internal standard is lower than the
threshold; and the decreasing rate of the ion intensity of ions
originated from the internal standard is employed as an error in the
reverse case. Similarly, when the ion enhancement occurs, the ion
intensity of ions originated from an internal standard increases. This
increasing rate can be included in the error as the lower limit of an
increasing rate in a measurement value of the ion intensity of other
components, and can be reflected to statistical processes of various
data. Provided that values of the isoelectric point and the dissociation
constant having being described herein are rough ones, and may involve an
error of about 10% by experience.

[0129] The descriptions hitherto have been on the premise of analyses
using a spray ionization method such as ESI, gas spray-assisted ESI and a
sonic spray ionization method (SSI), but in the case of APCI, the proton
affinity governing the gas-phase ion-molecule reaction is an important
physical quantity instead of the isoelectric point and the dissociation
constant. That is, setting the proton affinity of an internal standard
low can make the internal standard most susceptible to quantitative
analysis-inhibitory factors. For example, since the proton affinity of
amino acids and the like is 200 kcal/mol or more, components having a
lower proton affinity than the amino acids and the like, and a high
hydrophilicity are candidates of an internal standard. The utilization of
water (about 165 kcal/mol), methanol (about 180 kcal/mol), acetonitrile
(about 187 kcal/mol) or the like contained in an LC mobile phase solvent
is convenient. The decreasing rate of the ion intensity due to the
occurrence of quantitative analysis-inhibitory factors can be treated in
the same manner as in the case of ESI. Finally, a third requirement
condition of an internal standard is that m/z of the ions is different
from that of an object component for the analysis.

[0130]FIG. 12 shows an analysis example of a plasma sample. Total ion
chromatograms in the case where the injection amount was changed by two
digits from 0.5 μg to 0.005 μg are shown in (a) an overlapped
fashion. Mass chromatograms of ions originated from an internal standard
(DSSSSS) are shown in (b) an overlapped fashion. From this result, in the
case where the injection amount was 0.005 μg, a decrease in the
intensity of ions originated from the internal standard (DSSSSSS) is not
observed. Hence, the result is considered to be equal to the analysis
result of a blank sample, so this data for the analysis indicates no
problem even if a quantitation is performed. However, in the case where
the injection amount was 0.05 μg, the intensity of ions originated
from the internal standard decreased by about 20 to 40% after 50 min of
the retention time. Hence, other ion intensities also are considered to
possibly decrease to the same level after 50 min of the retention time.
In the case where the injection amount was 0.5 μg, the intensity of
ions originated from the internal standard outstandingly decreases over
the almost whole retention time region where separated components are
detected. In the example of FIG. 12, LC separation was performed in a
gradient mode. By contrast, in the case of performing LC separation in an
isocratic mode, the composition of a mobile phase is constant. Hence,
unless quantitative analysis-inhibitory factors occur, the ion intensity
of ions originated from an internal standard is constant. In such a case,
in a mass chromatogram of ions originated from an internal standard as
shown in FIG. 12(b), the ion intensity indicates entirely the same ion
intensity as a blank sample until a decrease of the ion intensity by
injection of a sample at about 12 min is observed. Therefore, in such a
case, there is no need for particularly saving the analysis result of the
blank sample. The average ion intensity in the early retention time
region can be treated in the same manner as the analysis result of the
blank sample. If this time region is present for 2 min or more, the
presence/absence of the occurrence of quantitative analysis-inhibitory
factors can be detected with high precision.

[0131] Comparison of mass spectra acquired at retention times indicated by
(1) and (2) in FIG. 12 is shown in FIG. 13 and FIG. 14, which indicate
that patterns of mass spectra acquired in the cases (c) where the
injection amount was maximum are different from the results of the cases
((a) and (b)) where the injection amount was low. This difference can be
interpreted as the occurrence of the ion suppression. Paying attention to
ions detected in FIG. 13 and FIG. 14 (m/z=637.97 and 588.96,
respectively), relations between areas of these ion peaks (obtained by
integrating peak areas in mass spectra in the retention time direction)
and the injection amounts are shown in FIG. 15.

[0132] Since this figure is double logarithm plots, the case where the
plots can be fit to a straight line of 1 in slant allows for a
quantitation. As shown in FIG. 15, for two types of ions described above,
in the cases where the injection amounts were 0.005 and 0.05 μg, the
data points can be plotted on a straight line of nearly 1 in slant, but
in the case where the injection amount was 0.5 μg, the data points are
clearly out of a straight line of 1 in slant. These ions in the case of
0.5 μg can be interpreted to undergo an outstanding ion suppression.
It is indicated that the deviation from the straight line is smaller than
the decrease of the intensity of ions originated from the internal
standard. This fact agrees with the interpretation that the decreasing
rate of the ion intensity becomes equal to or less than that of ions
originated from an internal standard.

[0133] In the case of performing the quantitation, ions for the detection
are processed as a data set made by collecting the retention time and m/z
(or m, or m and z) of the ions, the peak area or the ion intensity, and
the error of the peak area or the ion intensity. Including not only
errors in sample preparation and measurement errors in a mass
spectrometer but also effects of the ion suppression and ion enhancement
to the error of the peak area is considered to be important for the
improvement in precision of results of data analysis. Alternatively, ions
for the detection may be processed as a data set made by collecting the
retention time and m/z (or m, or m and z) of the ions, the peak area or
the ion intensity, and the presence/absence of the occurrence of
quantitative analysis-inhibitory factors. In this case, by excluding the
data in the retention time region where quantitative analysis-inhibitory
factors have occurred from the analysis, the precision of results of data
analysis can be expected to be held.

[0134] If a data set as described above is used, in comparison of a
plurality of samples, peak areas or ion intensities in data sets with
respect to ions in which the retention times and m/z (or m and z) are
each consistent can be compared and analyzed. Then, by taking into
consideration errors of peak areas or ion intensities included in data
sets, it becomes possible to analyze the presence/absence and the degree
of the variations with high precision.

[0135]FIG. 3 shows an example of displayed data in the display unit of
the data analysis unit. The flow of analysis steps in the Present Example
is shown in FIG. 9, and hereinafter, the analysis steps will be described
in detail using FIG. 3 and FIG. 9. FIG. 9 shows unit steps to analyze a
test specimen of either of a disease subject or a healthy subject.
Variant components having remarkable differences between the both may
have been extracted before the analysis of FIG. 9, and the variant
components only may be analyzed, or variant components may not be yet
specified, and each test specimen may be analyzed through the steps of
FIG. 9 for the purpose of analyzing all of constituting elements.

[0136]FIG. 3 is examples of the ion intensity 1201b (data b, black solid
line) of ions originated from an internal standard when a trypsin enzyme
digestive product of BSA (bovine serum albumin) was analyzed as a sample
for the analysis, and the ion intensity 1201a (data a, gray solid line)
of ions originated from the internal standard when a blank sample was
analyzed. As the internal standard, a synthetic peptide whose amino acid
sequence was SSSSSSK was used as in FIG. 2. As shown in FIG. 9, after the
start of analysis (S1001), first, mobile phases A and B and a mobile
phase C containing an internal standard are mixed in a predetermined
ratio (initial value), and introduced from the mobile-phase introduction
unit, and a blank sample is introduced from the sample introduction unit
nearly at the same time (S1002). The analysis is started with the timing
of the introduction of the blank sample set as time 0. Then, with the
mixing ratio of the mobile phase C held at a constant, while the mixing
ratio of the mobile phases A and B are varied in a predetermined
variation amount in terms of time, the mobile phases A and B are
introduced (S1003); the concentration of the internal standard is
controlled so as to be always constant; and sequentially, the separation
by LC (S1004), the ionization by the ionization unit (S1005), and the
mass spectrometry by the mass-analysis unit are performed, and the ion
intensity of ions originated from the internal standard, that is, data
(data a) of a mass chromatogram is acquired (S1006). After the mixing
ratio of the mobile phases is varied to a mixing ratio at the end over a
predetermined time, and data for the analysis are obtained, the analysis
is finished (S1007), and the data are saved in the data storage unit as
data a (S1008). By the steps above, the mass chromatogram (data a) of the
internal standard for reference acquired using the blank sample was
acquired (1050).

[0137] Then, a sample for the analysis is introduced, and mass
chromatogram data of ions originated from the internal standard is
similarly acquired. First, as in acquiring data a, the mixing ratio of
mobile phases A, B and C is set at the initial value, and those are
introduced, and the sample for the analysis is introduced nearly at the
same time (S1009). Then, with the mixing ratio of the mobile phase C
fixed at a constant, the mixing ratio of the mobile phases A and B is
varied in terms of time as in acquiring data a (S1010); and as in steps
1004 to 1006, sequentially, the separation (S1011) and the ionization
(S1012) are performed, and the mass spectrometry of ions for the
detection is performed to acquire data (data b) of a mass chromatogram of
ions originated from the internal standards (S1013). At this analysis,
while a real time analysis control is being performed (1051), data
analysis is performed (S1021) as described later. By the steps of the
above, the sample for the analysis is introduced, and the acquisition of
the mass chromatogram of ions originated from the internal standard and
the analysis of the sample are simultaneously performed (S1009 to S1023).

[0138] The electrospray ionization method (ESI) is used in the interface
of LC/MS, but ion intensities for every analysis are not always
consistent. Then, in the retention time region (the retention time region
I in FIG. 3) right before components which are contained in the sample
and are not separated are eluted, the ion intensity is normalized, that
is, a level adjustment is made. In the time region III in the figure, it
is indicated that the intensities of ions originated from the internal
standard are nearly consistent, and no quantitative analysis-inhibitory
factors occur in a broad range. By contrast, in the time region indicated
as the time region IV in FIG. 3, ion intensities are not consistent. It
can be interpreted that the occurrence of this inconsistency expresses
the occurrence of quantitative analysis-inhibitory factors such as the
ion suppression and a variation in the intensity of ions originated from
the internal standard at the time of acquiring data b (that is, at the
time of mixing the sample for the analysis). Data are processed such that
the data are normalized and compared so as not to be influenced by
high-frequency noise components and based on the average levels in each
retention time. For example, high-frequency components of the data are
eliminated, or otherwise, to extract low-frequency components, and data
and data in the same retention time are compared and calculated. For
example, in the normalization process, displaying is adjusted so that the
magnitude of data a is set 100%, and data b becomes 100%±5%. This
value, ±5%, is an example, and the value is saved in the control
system 8 in advance as a range acceptable for normalization (level
adjustment), or alternatively the system is configured such that an
operator can input the value. Also in the comparison process, nearly
similarly, data a is set 100%, and data b is calculated about how many
percents the data b is to data a. For example, when data b is 97% to data
a, it is judged that there is a difference of 3%. The numerical value
(here, 3%) determined in such a way is hereinafter referred to as a
difference ratio, and is defined as an index indicating the inconsistency
between data a and b.

[0139] Seeing the data in detail, it is found that there is an error
within several percents in data a and data b even in the time region III.
The error of several percents is well known to be a dispersion in
measurement of mass chromatograms by common LC/MS. Since there is an
error of the ion intensity in such a mass chromatogram, it is necessary
to judge that if a difference between data a and b is a minute deviation
within several percents coming from the measurement error of a
measurement apparatus, the deviation is accepted as a measurement
dispersion; and if a difference between data a and b exceeds a threshold,
the difference has occurred due to quantitative analysis-inhibitory
factors. Then, a difference ratio (inconsistency) threshold to become a
judgment criterion is determined in advance, and data are acquired and a
difference ratio (inconsistency) is calculated to compare with the
inconsistency threshold in real time. This inconsistency threshold is
saved in the control system 8 in advance as in the normalization
acceptable range as described above, or alternatively the system is
configured such that an operator can input. In the present Example, since
the measurement apparatus was assumed to exhibit an error in a regular
level, so had a measurement error of about 5 to 10%, the difference ratio
(inconsistency) threshold was set to be 15%. That is, the case where the
difference ratio d between data a and b exceeded 15% was judged to be due
to quantitative analysis-inhibitory factors. Such an error is considered
to depend on mass spectrometers, and the system is so configured that
errors according to the measurement apparatus can be input to the data
analysis unit 105. In the data analysis unit 105 in FIG. 1, level
adjustment means 114 to perform calculation and display control to
perform the level adjustment between data a and b as described above, and
calculation means 115 to calculate the inconsistency between data and to
compare with the inconsistency threshold saved in advance are built in.

[0140] The inconsistency detected of the time region IV in FIG. 3
indicates the occurrence of quantitative analysis-inhibitory factors such
as the ion suppression, and the precision of the data for the analysis of
the sample decreases in the time region IV, so the quantitation is
difficult. By contrast, the data in the time region III has a sufficient
precision, so the quantitation is possible. Then, by extracting only the
data of the sample for the analysis in the time region III, the
quantitative analysis of the sample for the analysis in the time region
III is performed. If the quantitative analysis of the sample for the
analysis can be sufficiently performed in this time region III, the
quantitation with a high reliability can be performed as it is. Thereby,
even in the case where quantitative analysis-inhibitory factors occur
during the analysis, data for a time region where the data is not
influenced by inhibitory factors can be effectively utilized, and the
waste of analysis can be eliminated. In the time region IV, since
quantitative evaluation-inhibitory factors occur to decrease the
precision of the quantitative evaluation result, and the data cannot be
used, as described later, the analysis mode may be switched so as to
preferentially perform the qualitative evaluation in the analysis real
time in the time region IV. In the case where data cannot be acquired
completely by one time of the analysis, the analysis of the above may be
repeated.

[0141] In the case where the occurrence of such quantitative
analysis-inhibitory factors such as the ion suppression is detected, by
subjecting a sample to concentration reduction, refinement, separation
and the like, the sample in which no quantitative analysis-inhibitory
factors occur can be prepared. Also in this case, as a result of putting
the sample under a feedback to reprepare the sample, unless an
inconsistency is observed in a mass chromatogram of ions originated from
the internal standard in the display unit of the data analysis unit, it
can be confirmed that quantitative analysis-inhibitory factors come not
to occur. Alterations of the preparation conditions of a sample are
repeatedly performed, and if a time region where no quantitative
analysis-inhibitory factors occur, or those occur but the quantitative
analysis can be performed without being influenced by the occurrence is
secured as a time region of a constant time length or longer, the
alterations of the preparation conditions of the sample may be controlled
to be stopped.

[0142]FIG. 4 shows an example in which quantitative analysis-inhibitory
factors remarkably occur. In the present Example, it is indicated that
the inconsistency in the ion intensities of ions originated from an
internal standard occurs in a very broad region shown as the time region
III in the figure and quantitative analysis-inhibitory factors occur, and
that the data are unsuitable for the quantitation. This case needs to
perform a reanalysis after performing countermeasures including: 1) the
amount of a sample for the analysis is reduced to about 1/10; 2)
separation and fractionation are performed in advance in the sample
preparation; and 3) ionic impurities are removed by desalting or the like
in the sample preparation. As described above depending on the
inconsistency of ion intensities, effective quantitative data for the
analysis cannot be acquired in some cases without countermeasures to the
sample. The control system was configured such that a standard value is
provided in advance in the times of the inconsistent region and the
consistent region of ion intensities, and if the time of the consistent
region of ion intensities is shorter than the standard time, the control
system controls the successive repreparation of the sample. The standard
time may be saved in the control unit 8 in advance, or may be input by an
operator.

[0143] In the retention time region indicated as a dashed-line ellipse in
FIG. 4, the intensity 1201b (data b) of ions originated from an internal
standard in the data for the analysis of a sample increases, and becomes
equal to that 1201a (data a) of a blank sample. However, examining mass
spectra in this case, ions different from ions originated from the
internal standard, but equal in m/z were detected. Therefore, also in
this retention time region, no quantitative analysis-inhibitory factors
cannot be said not to have occurred. As described herein, in the case of
analyzing a very complicated sample, ions almost coincide in m/z with
ions originated from an internal standard are observed in some cases, and
this fact may adversely affect the detection of quantitative
analysis-inhibitory factors. Then, mass chromatograms of ions originated
from an internal standard are compared in the real time of the analysis,
and if the inconsistency occurs, a tandem mass spectrometry such as MS/MS
is conveniently performed. This is because by examining a relation
between the ion intensity of ions originated from an internal standard
and the intensity of dissociated ions detected by a tandem mass
spectrometry in advance, and by acquiring tandem mass spectrometry
spectra in real time, the intrinsic ion intensity of ions originated from
the internal standard can be determined.

[0144] In the proteome analysis, the metabolome analysis and the marker
search, since components contained in a sample are not always known, both
of the qualitative (identification) analysis and the quantitative
(variation) analysis are necessary. However, the qualitative
(identification) analysis needs not only usual mass spectra, but also
acquiring tandem mass spectrometry spectra such as MS/MS in a high
throughput; and by contrast, the quantitative (variation) analysis does
not perform the tandem mass spectrometry as far as possible, and needs
acquiring usual mass spectra in a high throughput. That is, according to
the purposes, the priority order of data acquired by a mass spectrometer
is different.

[0145] Means was conventionally general in which for example, a
qualitative analysis was first performed, and after all components were
identified, a quantitative analysis was performed. Specifically, the
qualitative analysis of all components was performed by the following
process: a file to instruct the analysis procedure was stored in the
control system 108 of an analyzer; when the qualitative analysis was
performed, the control system 108 referred to the analysis procedure
instruction file; and for example, the following processes were repeated:
a usual mass spectrometry was performed once; thereafter, higher 10 peaks
of a spectrum were extracted and tandem analyzed; a usual mass
spectrometry was again performed to confirm the components; successively
higher 10 peaks of the spectrum were again extracted and tandem analyzed.
At this time, in the case where the types of components to be
qualitatively analyzed were few, for example, only about 15 types, such a
control was performed in some cases that after the qualitative analysis
of all the components were completed by the second time of the tandem
analysis, a quantitative analysis by a usual mass spectrometry was
performed. This procedure was in some cases referred to as the
qualitative analysis-preferential mode.

[0146] When the quantitative analysis was performed after such a
qualitative analysis was completed, the control system 108 referred to
another analysis procedure instruction file which had been stored in
advance in the control system 108, and a usual mass spectrometry was
performed to perform the quantitative analysis with high precision. That
is, it was a general procedure that respective analysis procedure
instruction files for the qualitative analysis and the quantitative
analysis (first and second analysis procedure instruction files) were
stored, and controls were performed according to these, and the analysis
was performed in the order of the qualitative analysis→the
quantitative analysis. However, since data acquisition for the
qualitative (identification) analysis was performed in advance, and
thereafter, data acquisition for the quantitative (variation) analysis
was performed, an analysis with high throughput could not be performed in
conformance with the conditions. By contrast, if there is a function to
change analysis modes in real time, in the case of analyzing a sample
containing multiple components, the function is advantageous from the
viewpoint of making the throughput high.

[0147] Then, as shown in FIG. 1 and FIG. 6, in the present invention, a
comparison was made between mass chromatograms of ions originated from an
internal standard in the real time of the analysis, and the control
system was configured so that in the case where an inconsistency had
occurred, the analysis mode was changed, for example, switched to the
qualitative (identification) analysis-preferential mode. That is, the
order of performing the qualitative/quantitative analyses is controlled
in real time according to data to be acquired. In the present Example,
for example, the analysis is performed in preference of the quantitative
analysis (quantitative analysis-preferential mode). Then, the
presence/absence of quantitative analysis-inhibitory factors are judged
by the above-mentioned means, and in the time region where the analysis
is not influenced by the inhibitory factors, the quantitative analysis is
continued, and data for the analysis is stored in the control system 108.
When the occurrence of inhibitory factors and the reveal of the influence
are detected, the analysis mode is changed in preference of the
qualitative analysis (qualitative analysis-preferential mode) because the
precision of the quantitative analysis conceivably decreased. Thereby,
even in the time region where the analysis was influenced by quantitative
analysis-inhibitory factors, the qualitative analysis was allowed to be
performed efficiently.

[0148] The above-mentioned quantitative analysis-preferential mode will be
described further in detail. Conventionally, a control not to perform the
qualitative analysis by the tandem was usually performed in the case of
the quantitative analysis mode. By contrast, in the present Example, a
control was performed so that the qualitative analysis could be anew
performed even during the quantitative analysis depending on the
characteristics of a spectrum. This is referred to as the quantitative
analysis-preferential mode. For example, also in the time region where
quantitative analysis-inhibitory factors are not detected, components to
be presumed in advance to be present in a sample for the analysis are
stored in the control system 108, and if a spectrum of a component to
have not been presumed is acquired, the qualitative analysis may be
performed during the quantitative analysis. Utilizing this function, a
component analysis result of a sample for the analysis, for example, from
a test specimen of a healthy subject is acquired and stored in the
control system 108; and when a test specimen of a disease subject is
analyzed, the result is referred to, and the analysis of only a component
which has not been detected in the specimen from the healthy subject is
switched to the qualitative analysis, thus enabling identification of the
component. By controlling the analysis procedure in such a manner, the
precision and the efficiency (that is, throughput) both have been
remarkably improved in marker searches and the like.

[0149] In this case, the internal standard may be one type, or may be two
types as described later. As described in the figure, the analyzer was
configured so that the screen of the data analysis unit, or the screen of
the control unit of the mass spectrometer displayed the state of the
analysis as "quantitative analysis (preferential) mode", "qualitative
analysis (preferential) mode", or the like in real time, and that an
operator could confirm visually that the mass spectrometer operated
normally. The mode switching may be controlled such that something like a
third analysis procedure instruction file is stored in the control system
108, and "the qualitative analysis-preferential mode" and "the
quantitative analysis-preferential mode" are switched based on data
successively acquired, or may be performed by a constitution in which as
shown in FIG. 6, the display unit has a mode selection button 109 of the
qualitative/quantitative analyses, and an operator switches the modes
with a pointer 110 by a pointing device 112 or the like according to
needs while the operator monitors successively the analysis results. The
judgment criterion to judge the switching may be stored in the control
unit 8, or may be input by an operator.

[0150] As described hitherto, the control system for the control
represented as real-time analysis control (1051) in FIG. 9 was configured
to involve: (1) the normalization and level adjustment in real time of
data a and data b in the analysis initial stage (S1014, S1015); (2) the
judgment of the presence/absence of real-time quantitative
analysis-inhibitory factors (the inconsistency between intensity data a
and b of ions originated from an internal standard) (S1016) and the data
extraction in an effective time region where no quantitative
analysis-inhibitory factors occur by the result of the judgment (S1017);
(3) the modification of conditions for separating and preparing samples,
and the like (S1018, S1019), in the case where the degree of the
influence of quantitative analysis-inhibitory factors (a length of time
where data a and b are consistent, and the like) is large (the length of
time of the consistency is equal to or smaller than a standard value);
and (4) the switching and selection of the preferential mode of the
quantitative/qualitative (tandem) analyses in the time region where the
analysis is influenced by quantitative analysis-inhibitory factors
(S1020) to perform the quantitative and qualitative analyses by analyzing
data acquired (S1021). Further for example, also in the quantitative
analysis-preferential mode, in the case where an outstanding
characteristic was found in the spectrum, for example, a peak of a
component not expected was detected as compared with an expected
component of a sample for the analysis stored in advance, the analysis
mode was controlled in real time to perform the qualitative analysis. In
the case where an analysis is finished and data for the analysis have not
been acquired completely, the analysis is repeated to acquire all data
required.

[0151] In the data analysis unit 105, means 116 to detect a time region
where the inconsistency is smaller or larger than a threshold of the
inconsistency, and means 117 to collect data for the analysis in a time
region where the inconsistency is small, that is, the consistency is
large (time region of consistency) are built in. The data analysis unit
has a constitution having a storage unit for the above-mentioned analysis
procedure instruction file, and a control unit to read the file.

EXAMPLE 2

[0152] Next, as Second Example, analysis means using two types of internal
standards will be described using FIG. 5 and FIG. 10. As shown in FIG.
10, use of two types of internal standards does not allow for evaluation
by performing two times of analyses as in Example 1, but allows for
evaluation of the presence/absence of the occurrence of quantitative
analysis-inhibitory factors by one time of the analysis. Thereby, the
analysis time can be further reduced. The two types of internal standards
are selected such that these are substances to be always detected as
ions, that is, hydrophilic substances; and one of them is acidic, and the
other thereof is basic; and the former sensitively changes to
quantitative analysis-inhibitory factors, and the latter hardly changes.

[0153] That is, one of the internal standards to be selected has an
isoelectric point of about 3 or more and 8 or less; and the other thereof
has that nearly equal to or more than 8. Here, a substance having a high
hydrophilicity and a high acidity is denoted as a first internal
standard; and a substance having a high hydrophilicity and a high
basicity is denoted as a second internal standard. The upper part of FIG.
5 shows a mass chromatogram (1202a, black solid line) of ions originated
from the first internal standard, and a mass chromatogram (1202b, gray
dashed line) of ions originated from the second internal standard
obtained in the present Example. In the present Example, in the time
region I in FIG. 5, the level adjustment of the intensities and the
normalization of the intensities of ions originated from the first and
second internal standards are performed in real time, and the ion
intensities are overlappingly displayed in the display unit of the data
analysis unit. In analysis by LC/MS, there was a possibility of causing
an unexpected subtle variation in the ion amount caused by the analyzer,
but in the case of performing the analyses two times as in Example 1,
since random variations may occur every time, the influence of the
unexpected random variations in the ion amount cannot be avoided.
However, in the present Example, since two data can simultaneously be
acquired and compared, even when an unexpected variation in the ion
amount is caused due to the analyzer, similar variations occur in the
both data and the difference between the both ends in not being affected
by the ion variation. Consequently, the present Example has an advantage
in which the evaluation of the inconsistency can be performed more
precisely and accurately than Example 1.

[0154] The lower part of FIG. 5 is a total ion chromatogram, and data for
the analysis in the retention time region (time region III) different
from the retention time (time region IV) when quantitative
analysis-inhibitory factors occur can be subjected to the quantitation.
Then, only the data for the analysis in the retention time region (time
region III) is extracted, and stored in the data analysis unit or the
like under another name. In such a manner, only data for the analysis
allowing to be subjected to the quantitation can be analyzed. As
described above, the present Example is different from First Example in
the point that while the presence/absence of the occurrence of
quantitative analysis-inhibitory factors is being judged by one time of
the analysis using two types of internal standards, the target sample for
the analysis is analyzed; and other points are the same as in First
Example. That is, the inconsistency between two internal standards is
calculated from mass chromatograms thereof as in First Example; the
inconsistency is compared with a threshold of the inconsistency
determined in advance; an analysis time region where the inconsistency is
smaller than the threshold is detected; and data for the analysis in this
analysis time region is collected. The two internal standards are
injected in a constant concentration to a mobile phase, and the analysis
is configured so that these can be detected stably over the whole
analysis time.

[0155] As in First Example, in the case where the inconsistency between
the two mass chromatograms are large, the
quantitative/qualitative-analysis preferential modes are switched and are
put in preference of the qualitative analysis (tandem analysis), and the
switching situations are displayed; and in the case where the length of
the time region when the inconsistency becomes sufficiently small is
shorter than the standard time determined in advance, the preparation
condition of the sample is modified and a control is performed so as to
repeat the preparations until the inconsistency becomes small to achieve
the analysis with high efficiency and high precision. The constitution of
the analyzer used in Second Example is nearly the same as that used in
First Example shown in FIG. 1, except that details of calculation
contents of the data analysis unit 105 and calculation means in the
control unit 8 are partially different.

EXAMPLE 3

[0156] As Third Example, means will be described in which one type of an
internal standard is introduced and denoted as a first internal standard;
and a second internal standard is not positively introduced, and a
component capable of becoming a second internal standard is searched from
component substances such as impurities unintentionally mixed; and mass
chromatograms of the both are simultaneously compared. The analysis steps
of the present Example, as shown in FIG. 11, has a step (S1024) of
searching and selecting a substance usable as a second internal standard,
the step being added as compared to Second Example in FIG. 10. In this
case, the first internal standard to be selected is, as in the first
internal standard in Second Example, a hydrophilic and acidic substance,
and the substance sensitively reacting to quantitative
analysis-inhibitory factors.

[0157] A search monitor for the second internal standard is provided; and
a substance which is present in the analyzer and stably detected as ions
in a broad retention time region, and additionally little influenced by
quantitative analysis-inhibitory factors is detected by changing mobile
phases and samples. For example, ions of a type of impurities such as
siloxane were observed stably in a broad retention time region, and
additionally little varied in the ion intensity to quantitative
analysis-inhibitory factors such as the ion suppression. Such a substance
is selected as the second internal standard; and mass chromatograms of
the first and second internal standards are simultaneously acquired and
compared as in Second Example to detect the quantitative
analysis-inhibitory factors. Other parts of the procedure are the same as
in First and Second Examples. The analyzer of the present Example has, as
compared with the analyzer constitution of First Example, a constitution
further concurrently having monitoring means to monitor data for the
analysis of a plurality of substances in order to search a substance
capable of becoming a second internal standard, and an input unit to
select and input the searched and found substance as the second internal
standard, that is, an input device such as a pointing device or a key
board, a selection menu and a selection button, a display menu such as a
numerical value input column, and the like.

EXAMPLE 4

[0158] Then, as Fourth Example, means will be described in which a type of
an internal standard is introduced; and quantitative analysis-inhibitory
factors are detected by only the analysis result by introduction of a
sample for the analysis without an analysis of a blank sample. As shown
in FIG. 2, the mass chromatogram of the internal standard has a tendency
of not rapidly varying in terms of time. Hence, without using an analysis
result of a blank sample, it is possible in principle to detect the
occurrence of quantitative analysis-inhibitory factors. Then, only in the
condition that the sample for the analysis is introduced, by the
acquisition of a mass chromatogram of ions originated from the internal
standard, and the examination of whether or not there is a rapid change
in terms of time therein, the quantitative analysis-inhibitory factors
are detected. This analysis means, in the case where the decrease in the
intensity of ions originated from the internal standard due to
analysis-inhibitory factors is not rapid, needs to be paid attention to
in the point that it becomes difficult to detect the occurrence of the
analysis-inhibitory factors.

EXAMPLE 5

[0159] In a constitution diagram of another example in a mass spectrometry
system according to the present invention shown in FIG. 7, unlike the
example of FIG. 1, an internal standard 204 is injected to the downstream
of the separation unit 103 from a syringe pump 111. Thereby, the internal
standard 204 is mixed with an eluate of the liquid chromatograph in a
constant ratio. Of course, it suffices if the internal standard 204 can
be mixed with a liquid for the analysis, and the mixing place may be
anywhere as long as the upstream side of the interface (ion source). In
the case of using a conventional liquid chromatograph, a semi-micro
liquid chromatograph and a micro liquid chromatograph, whose flow rates
are higher than 1 microliter/min, such a constitution is more
advantageous than the constitution shown in FIG. 1. This is because the
exchange of a solution containing the internal standard is easy. The
present Example can be performed in combination with First to Fourth
Examples.

EXAMPLE 6

[0160]FIG. 8 shows a retention time dependency of a mass shift utilizing
the detection of ions originated from the internal standard in another
example of a mass spectrometry system according to the present invention.
If the analysis using a liquid chromatograph takes several or more
minutes, the precision in mass in a ppm level decreases due to the
temperature change and the like in some cases. Then, by examining the
measurement value (1402 in FIG. 8) of m/z of ions originated from an
internal standard having a known calculated mass (1401 in FIG. 8) with
respect to the retention time, the measurement value (1404 in FIG. 8) of
m/z of ions detected at each retention time can be corrected by the
proportional distribution (1403 in FIG. 8). Hence, particularly in the
data base retrieval in the qualitative analysis, the identification of a
substance can be performed with very high precision. The present Example
can be performed in combination with First to Fifth Examples.

[0161] Hitherto, means to detect quantitative analysis-inhibitory factors
for the field of marker searches mainly for disease diagnoses, and mass
spectrometry systems utilizing the means have been described. On the
other hand, in the field such as pharmacokinetics using a tandem mass
spectrometry referred to as Multiple Reaction Monitoring: MRM, the tandem
mass spectrometry is used not only for the qualitative analysis for a
substance but also for the quantitative analysis. Such a case needs that
a tandem mass spectrometry of ions originated from an internal standard
is performed in advance, and m/z of one type or several types of major
fragment ions are registered in a mass spectrometer. Thereby, a mass
chromatogram of major fragments of ions originated from an internal
standard can be acquired. Then, in the mass chromatogram of the fragment
ions, the occurrence of quantitative analysis-inhibitory factors can be
detected by the variation (decrease) of the intensity. The specific
method for the detection and the like are the same as those described
hitherto.

EXAMPLE 7

[0162] The technology according to the present invention can be applied to
automatic analyzers and diagnosing apparatuses to examine the
concentrations and the amounts of drugs and the like in blood and urine.
Then, hereinafter, particularly an example of a constitution and steps of
an automatic analyzer using the solid-phase extraction method will be
described. In the sample preparation for an automatic analyzer and a
diagnosing apparatus, a different method may be used other than a
solid-phase extraction method and a method for introducing a sample to
the mass analysis unit described in the present Example, but the effect
can be exhibited similarly.

[0163] An automatic analyzer in the present Example is, as shown in FIG.
16 and FIG. 17, constituted of a solid-phase extraction unit (16A), a
detection unit (16B), and a control unit (16C). In the example, a storage
unit is contained in the control unit.

[0164] The solid-phase extraction unit (16A) is equipped with a turn table
301 on which cartridge-holding containers 303 to hold disposable
solid-phase extraction cartridges 302 are disposed, a cartridge storage
unit 312 to store the solid-phase extraction cartridges 302, a rotary arm
309 to move the solid-phase extraction cartridges 302 from the cartridge
storage unit 312 to the cartridge-holding containers 303, a turn
table-type reagent tank 310 in which reagent containers 311 are disposed,
a rotary arm 308 to transport reagents from the reagent containers 311 to
the solid-phase extraction cartridges 302, a pressure loading unit 304 to
perform the extraction step by loading a pressure on at least one
solid-phase extraction cartridge 302, a turn table 305 on which a
plurality of receiving containers 306 to receive solutions extracted from
the solid-phase extraction cartridges 302 is disposed under the turn
table 301, the rotary arm 308 to transport the extracted solutions from
the receiving containers 306 to a sample introduction unit 316, and a
liquid surface sensor 307 to detect the degree of progress of the
extraction.

[0165] The solid-phase extraction cartridge 302 is equipped with a
pressure releasing valve to operate to release the pressure, and the
constitution is such that the pressure releasing valve is released when
the liquid surface detected by the liquid surface sensor reaches the
liquid surface position previously set.

[0166] The detection unit (16B) is equipped with a pump 315 to extrude the
solution in order to introduce a sample to an ionization unit, the
ionization unit 317 to ionize the sample by impressing a voltage, a
sample introduction unit 316 located at the post-stage of the pump 315
and the pre-stage of the ionization unit 317 and to introduce the sample
into a flow passage, and a mass-analysis unit 318 to analyze/examine the
ionized sample.

[0167] The control unit (16C) is composed of a control unit 319 to control
automatically and collectively each unit constituting the analyzer.

[0168] Hereinafter, the examination/analysis of the analyzer including
solid-phase extraction operation will be described in the order of steps.

Standard Reagent Addition Step

[0169] A standard reagent in a constant concentration is added to a sample
transported by the sample transportation unit 313. The addition is
performed such that the standard reagent in the reagent container 311 in
the reagent tank 310 is sucked by the rotary arm 308, and the reagent is
added to the sample transportation unit 313. As the standard reagent,
desirable is use of a stable isotope-labeled molecule obtained by
substituting hydrogen (H) or carbon (C) of drugs or the like being an
object of the examination/analysis contained in the sample with 2H
or 13C. However, in the case where availability of the stable
isotope-labeled molecule is difficult, a chemical analog whose chemical
structure is partially different from the object substance for the
analysis (drug or the like) is used. Although it is desirable that the
chemical analog is the same as the object substance for the analysis in
physicochemical properties as is the case with the stable isotope-labeled
molecule, there is no guarantee therefor. The ends of the rotary arms
308, 309 and 314 are each equipped with a pipette or a syringe to
suck/discharge the reagent, and with a mechanism to automatically
cleaning the end after suction discharge of the reagent.

Attachment and Detachment of the Solid-Phase Extraction Cartridge 302

[0170] The cartridge storage unit 312 is arranged in the turn table 301
with the same angles from the center; and the solid-phase extraction
cartridges 302 are replaceable, and successively transported by the
rotary arm 309 and installed in the cartridge-holding containers 303. The
solid-phase extraction cartridges 302 are installed in the
cartridge-holding containers 303 by transport means such as a belt
conveyer in some cases.

Cleaning Step of the Solid-Phase Extraction Cartridge 302

[0171] Then, the solid-phase extraction cartridge 302 is cleaned. The
cleaning step is such that the turn table 301 rotates to the operational
range of the rotary arm 308; a reagent for cleaning in the reagent
container 311 in the reagent tank 310 is sucked by the rotary arm 308;
and the reagent for cleaning is injected to the solid-phase extraction
cartridge 302. Then, the turn table 301 rotates to the operational range
of the pressure loading unit 304; and a pressure is loaded to move the
reagent for cleaning from the upper part to the lower part of the
solid-phase extraction cartridge 302 to perform the cleaning step. As
shown in FIG. 17, the turn table 305 having the same shape as the turn
table 301 is arranged vertically under the turn table 301; in the case
where a component for the extraction is necessary to be captured, the
receiving container 306 is arranged vertically under the
cartridge-holding container 303 to capture the component for the
extraction, by the rotary angles of the turn table 301 and the turn table
305. In the case where there is no need for the capture of the component
for the extraction, the eluted component is disposed of as a waste
liquid. The turn table 301 and the turn table 305 have a mechanism
capable of rotating them to the clockwise rotary direction and the
anticlockwise rotary direction, and can rotate to the direction in which
they can move to the next operational position in a short time.

[0172] In the cartridge-holding container 303 of the turn table 301, the
plurality of solid-phase extraction cartridges 302 is arranged; and the
suction and injection operations of the reagent, and the loading
operation of a pressure can be simultaneously performed for each
solid-phase extraction cartridge 302.

[0173] With respect to the relation between the shape of the turn table
301 and the positions of the cartridge-holding containers 303, the
cartridge-holding containers 303 are positioned evenly with the same
angles from the center of the circular turn table 301.

[0174] The relation between the shapes and the positions of the
cartridge-holding containers 303 arranged on the turn table 301 and the
receiving containers 306 arranged on the turn table 305 can assume the
following structures. That is, the turn table 301 and the turn table 305
have the same shape; and the cartridge-holding containers 303 and the
receiving containers 306 correspond to each other one to one in the
vertical directions. Alternatively, the turn table 301 and the turn table
305 have the same shape; but the cartridge-holding containers 303 and the
receiving containers 306 do not correspond to each other one to one, and
the shape may be such that one cartridge-holding container 303 has a
plurality of receiving containers 306. Further alternatively, the turn
table 301 and the turn table 305 have different shapes, for example, an
elliptic shape or a linear shape, and the shape may be such that one
cartridge-holding container 303 has a plurality of receiving containers
306 according to the different shapes.

Equilibration Step to the Solid-Phase Extraction Cartridge 302

[0175] The solid-phase extraction cartridge 302 once cleaned with an
organic solvent is subjected to the equilibration so that a drug
component in the sample becomes in the state capable of being adsorbed in
the solid-phase extraction cartridge 302. The equilibration step is such
that the reagent tank 310 rotates to the operational range of the rotary
arm 308; and a reagent for the equilibration in the reagent container 311
is sucked and discharged by the rotary arm 308, and injected into the
solid-phase cartridge 302. Then, the turn table 301 rotates to the
operational range of the pressure loading unit 304; and a pressure is
loaded to move the reagent for the equilibration from the upper part to
the lower part of the solid-phase extraction cartridge 302, thereby
performing the equilibration step. The reagent for the equilibration to
be used is usually an aqueous solution.

Adsorption Step to the Solid-Phase Extraction Cartridge 302

[0176] A sample to which a standard reagent in a constant concentration
has been added is injected to the solid-phase extraction cartridge 302
having being subjected to the equilibration to adsorb the drug component
in the sample. The adsorption step is such that the sample transportation
unit 313 rotates to the operational range of the rotary arm 314; and the
sample on the sample transportation unit 313 is sucked/discharged by the
rotary arm 314, and injected to the solid-phase cartridge 302. Then, the
turn table 301 rotates to the operational range of the pressure loading
unit 304; and a pressure is loaded to move the reagent for the
equilibration from the upper part to the lower part of the solid-phase
extraction cartridge 302, thereby performing the adsorption step.

Cleaning Step

[0177] By performing the cleaning step, nonspecifically adsorbed
components among components adsorbed on the solid-phase extraction
cartridge 302 in the adsorption step leave the solid-phase extraction
cartridge 302, thereby concentrating the target drug component. The
cleaning step is such that the reagent tank 310 rotates to the
operational range of the rotary arm 308; and a reagent for cleaning in
the reagent container 311 is sucked/discharged by the rotary arm 308, and
injected to the solid-phase cartridge 302. Then, the turn table 301
rotates to the operational range of the pressure loading unit 304; and a
pressure is loaded to move the reagent for cleaning from the upper part
to the lower part of the solid-phase extraction cartridge 302, thereby
performing the cleaning step. The reagent for cleaning to be used is
usually a solution containing mainly an organic solvent such as methanol
or acetonitrile.

Elution Step

[0178] The drug component adsorbed on the solid-phase extraction cartridge
302 is eluted. The elution step is such that a reagent for the elution is
injected to the solid-phase extraction cartridge 302 as in the cleaning
step; and a pressure is loaded to move the reagent for the elution from
the upper part to the lower part of the solid-phase extraction cartridge
302, thereby performing the elution step. The reagent for the elution
contains an internal standard in a constant concentration, and as a
solvent, an organic solvent such as methanol or acetonitrile is used.

Introduction to the Detection Unit

[0179] The eluted solution is introduced to the detection unit (16B) to
perform the examination/analysis. The introduction to the detection unit
(16B) is made such that the turn table 305 rotates to the operational
range of the rotary arm 308, and the eluted solution is sucked/discharged
from the receiving container 306, and introduced to the sample
introduction unit 316. In the ionization unit 317, the ionization is
performed by the electrospray ionization method (ESI) or an atmospheric
pressure chemical ionization method (APCI). For the ionization unit, the
matrix assisted laser desorption ionization method (MALDI) also is
conceivable which performs the ionization by a MALDI plate and the
irradiation of a laser light.

[0180] The object substance for the analysis, its standard reagent (stable
isotope-labeled molecule or a chemical analog) and the internal standard
ionized in the ionization unit are subjected to the mass separation and
the detection by the mass-analysis unit 318. Then, the intensities of
ions originated from them are determined, respectively.

Evaluation of Acquired Data in the Control Unit

[0181] If the intensity of ions originated from the internal standard is
consistent with that of the blank sample within the threshold range, no
ion suppression is confirmed to have occurred. In this case, in the
control unit, the concentration and amount of the object substance for
the analysis can be determined and output using a calibration curve from
the intensity of ions originated from the object substance for the
analysis, based on the intensity of ions originated from the standard
reagent.

[0182] On the other hand, unless the intensity of ions originated from the
internal standard is consistent with that of the blank sample within the
threshold, the ion suppression is confirmed to have occurred. Although
the quantitation has no problem in the case where the standard regent is
a stable isotope-labeled molecule, in the case where the standard reagent
is a chemical analog, it is desirable that the pretreatment condition and
the like are partially changed and the reanalysis is performed. An
example of changing the pretreatment condition conceivably involves an
increase in the amount of the reagent for cleaning to be injected to the
solid-phase extraction cartridge in the cleaning step. As a result of the
reanalysis, if the intensity of ions originated from the internal
standard is consistent with that of the blank sample within the
threshold, the concentration and amount of the object substance for the
analysis can be determined and output from the intensity of ions
originated from the object substance for the analysis, based on the
intensity of ions originated from the standard reagent. Unless the
intensity of ions originated from the internal standard is consistent
with that of the blank sample within the threshold, the pretreatment
condition is further changed partially, and the reanalysis is performed.
Performing such a reanalysis results in a temporarily decreased analysis
throughput. However, unless reanalyses frequently occur, there is no
problem in practical use. In the information of the data acquired in the
reanalysis, the information on changed analysis conditions is desirably
contained. In the analysis condition to be changed, a calibration curve
is desirably obtained in advance.

[0183] In the case where the standard reagent is a chemical analog and the
execution of the reanalysis as described above is difficult, it is
practical that the concentration and amount of the object substance for
the analysis are presumed (corrected) and output from the intensity of
ions originated from the object substance for the analysis, based on the
intensity of ions originated from the standard reagent. The presumption
conceivably includes a method in which the occurrence of the ion
suppression is taken into account, and other various methods. However,
since in the decreasing rate of the ion intensity caused by the
occurrence of the ion suppression, a difference can be caused between the
object substances for the analysis and the chemical analog, the presumed
value is significantly different from a true value in some cases. Then,
it is considered to be effective that an error reflecting the decreasing
rate of ions originated from the internal standard is imparted to the
presumed value.

[0184] Hereinafter, a simple presumption example will be considered. That
is, the decreasing rate of ions originated from a chemical analog caused
by the occurrence of the ion suppression is denoted as T [%]; the
intensity of ions originated from the chemical analog is converted to
100/(100-T) times; and the concentration and amount of the object
substance for the analysis is presumed from the ratio of the converted
ion intensity and the intensity of ions originated from the object
substance for the analysis. At this time, there is a possibility that
ions originated from the object substance for the analysis are not at all
influenced by the ion suppression, whereas there is also a possibility
that the ion intensity is decreased as largely as ions originated from
the internal standard. That is, if the decreasing rate of ions originated
from the internal standard is denoted as Tp [%], there is a
possibility that a presumed value is corrected excessively by T [%],
whereas there is also a possibility that the correction is insufficient
by 100{1-(100-Tp)/(100-T)}[%]. Then, by reflecting these to the
error information of the presumed value, the difference from a true value
can be expressed. Thus, to impart the error information based on the
decreasing rate of the intensity of ions originated from a chemical
analog and the decreasing rate of ions originated from an internal
standard to the error information of the presumed value connects directly
with the maintenance of a high reliability in data obtained by automatic
analyzers and diagnosing apparatuses. Of course, as the presumption
method of the intensity of ions originated from an object substance for
the analysis based on the intensity of ions originated from a chemical
analog, another method may be employed. It is important that the error
information based on the decreasing rate of the intensity of ions
originated from a chemical analog and the decreasing rate of ions
originated from an internal standard is reflected to the error
information of a presumed value.

EXAMPLE 8

[0185] In automatic analyzers and diagnosing apparatuses to examine the
concentrations and amounts of drugs and the like in blood and urine, a
method for preparing samples without using the solid-phase extraction
method can be employed. For example, a solution for the analysis is
diluted to such a degree that no ion suppression is expected to occur,
and a high-efficient ionization is performed at a low flow rate of
several nano-liters/min by the electrospray ionization method
(nanoelectrospray ionization method), which is effective. Hereinafter, an
example of the analysis procedure will be described.

[0186] First, only a dilute solution containing an internal standard and a
standard reagent in constant concentrations are filled in a chip for a
nano-spray whose tip end is in a micron size, to make a blank sample for
the analysis. Thereby, a reference data is acquired. Then, a solution for
the analysis is diluted with the diluted solution described above, and
filled in a chip for another nano-spray, and analyzed. The result is
compared with the reference data, and if the ion intensity of ions
originated from the internal standard is consistent within the threshold
range, no occurrence of the ion suppression is confirmed. In this case,
the concentration and amount of the object substance for the analysis can
be determined from the ratio of the intensities of ions originated from
the object substance for the analysis to ions originated from the
standard reagent. By contrast, unless the ion intensity of ions
originated from the internal standard is consistent within the threshold
range, the inconsistency can be reflected to the error of the measurement
value. However, in order to obtain measurement values with high
precision, a remeasurement needs to be performed by increasing the
dilution magnification of the solution for the analysis. Such a
remeasurement is desirably automatically performed in the analyzers and
the diagnosing apparatus.

[0187] All of publications, patents, and patent applications referred to
in the present description are incorporated into the present description
as they are herein by standard.